Assays and reagents for identifying anti-fungal agnets, amd uses related thereto

ABSTRACT

The present invention relates to rapid, reliable and effective assays for screening and identifying pharmaceutically effective compounds that specifically inhibit the biological activity of fungal GTPase proteins, particularly GTPases involved in cell wall integrity, hyphael formation, and/or other cellular functions critical to pathogenesis.

GOVERNMENT FUNDING

[0001] Work described herein was supported in part by funding from theNational Institute of Health. The United States Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

[0002] Fungal infections of humans range from superficial conditions,usually caused by dermatophytes or Candida species, that affect the skin(such as dermatophytoses) to deeply invasive and often lethal infections(such as candidiasis and cryptococcosis). Pathogenic fungi occurworldwide, although particular species may predominate in certaingeographic areas.

[0003] In the past 20 years, fungal infections have increaseddramatically—along with the numbers of potentially invasive species.Indeed, fungal infections, once dismissed as a nuisance, have begun tospread so widely that they are becoming a major concern in hospitals andhealth departments. Fungal infections occur more frequently in peoplewhose immune system is suppressed (because of organ transplantation,cancer chemotherapy, or the human immunodeficiency virus), who have beentreated with broad-spectrum antibacterial agents, or who have beensubject to invasive procedures (catheters and prosthetic devices, forexample). Fungal infections are now important causes of morbidity andmortality of hospitalized patients: the frequency of invasivecandidiasis has increased tenfold to become the fourth most common bloodculture isolate (Pannuti et al. (1992) Cancer 69:2653). Invasivepulmonary aspergillosis is a leading cause of mortality in bone-marrowtransplant recipients (Pannuti et al., supra), while Pneumocystiscarinii pneumonia is the cause of death in many patients with acquiredimmunodeficiency syndrome in North America and Europe (Hughes (1991)Pediatr Infect. Dis J. 10:391). Many opportunistic fungal infectionscannot be diagnosed by usual blood culture and must be treatedempirically in severely immunocompromised patients (Walsh et al. (1991)Rev. Infect. Dis. 13:496).

[0004] The fungi responsible for life-threatening infections includeCandida species (mainly Candida albicans, followed by Candidatropicalis), Aspergillus species, Cryptococcus neoforms, Histoplasmacapsulatum, Coccidioides immitis, Pneumocystis carinii and somezygomycetes. Treatment of deeply invasive fungal infections has laggedbehind bacterial chemotherapy.

[0005] There are numerous commentators who have speculated on thisapparent neglect. See, for example, Georgopapadakou et al. (1994)Science 264:371. First, like mammalian cells, fungi are eukaryotes andthus agents that inhibit fungal protein, RNA, or DNA biosynthesis may dothe same in the patient's own cells, producing toxic side effects.Second, life-threatening fungal infections were thought, until recently,to be too infrequent to warrant aggressive research by thepharmaceutical industry. Other factors have included:

[0006] (i) Lack of drugs. A drug known as Amphotericin B has become themainstay of therapy for fungal infection despite side effects so severethat the drug is known as “amphoterrible” by patients. Only a fewsecond-tier drugs exist.

[0007] (ii) Increasing resistance. Long-term treatment of oralcandidiasis in AIDS patients has begun to breed species resistant toolder anti-fungal drugs. Several other species of fungi have also begunto exhibit resistance.

[0008] (iii) A growing list of pathogens. Species of fungi that onceposed no threat to humans are now being detected as a cause of diseasein immune-deficient people. Even low-virulence baker's yeast, found inthe human mouth, has been found to cause infection in susceptible burnpatients.

[0009] (iv) Lagging research. Because pathogenic fungi are difficult toculture, and because many of them do not reproduce sexually,microbiological and genetic research into the disease-causing organismshas lagged far behind research into other organisms.

[0010] In the past decade, however, more antifungal drugs have becomeavailable. Nevertheless, there are still major weaknesses in theirspectra, potency, safety, and pharmacokinetic properties, andaccordingly it is desirable to improve the the panel of anti-fungalagents available to the practioner.

[0011] I. The Fungal Cell

[0012] The fungal cell wall is a structure that is both essential forthe fungus and absent from mammalian cells, and consequently may be anideal target for antifungal agents. Inhibitors of the biosynthesis oftwo important cell wall components, glucan and chitin, already exist.Polyoxins and the structurally related nikkomycins (both consist of apyrimidine nucleoside linked to a peptide moiety) inhibit chitinsynthase competitively, presumably acting as analogs of the substrateuridine diphosphate (UDP)-N-acetylglucosamine (chitin is anN-acetylglucosamine homopolymer), causing inhibition of septation andosmotic lysis. Unfortunately, the target of polyoxins and nikkomycins isin the inner leaflet of the plasma membrane; they are taken up by adipeptide permease, and thus peptides in body fluids antagonize theirtransport.

[0013] In most fungi, glucans are the major components that strengthenthe cell wall. The glucosyl units within these glucans are arranged aslong coiling chains of β-(1,3)-linked residues, with occasionalsidechains that involve β-(1,6) linages. Three β-(1,3) chains running inparallel can associate to form a triple helix, and the aggregation ofhelicies produces a network of water-insoluble fibrils. Even in thechitin-rich filamentous aspergilli, β-(1,3)-glucan is required tomaintain the integrity and form of the cell wall (Kurtz et al. (1994)Antimicrob Agents Chemother 38:1408-1489), and, in P. carinii, it isimportant during the life cycle as a constituent of the cyst (ascus)wall (Nollstadt et al. (1994) Antimicrob Agents Chemother 38:2258-2265.

[0014] In a wide variety of fungi, β-(1,3)-glucan is produced by asynthase composed of at least two subunits (Tkacz, J. S. (1992) inEmerging Targets in Antibacterial and Antifungal Chemotherapy (Sutcliffeand Georgopapadakou, eds), pp495-523, Chapman & Hall; and Kang et al.(1986) PNAS 83:5808-5812). One subunit is localized to the plasmamembrane and is thought to be the catalytic subunit, while the secondsubunit binds GTP and associates with and activates the catalyticsubunit (Mol et al. (1994) J Biol Chem 269:31267-31274).

[0015] Two groups of anticandidal antibiotics known in the art interferewith the formation of β-(1,3)-glucan: the papulacandins and theechinocandins (Hector et al. (1993) Clin Microbiol Rev 6:1-21). However,many of the papulacandins are not active against a variety of Candidaspecies, or other pathogenic fungi including aspergillus. Theechinocandins, in addition to suffering from narrow activity spectrum,are not in wide use because of lack of bioavilability and toxicity.

[0016] II. Protein Prenylation

[0017] Covalent modification by isoprenoid lipids (prenylation)contributes to membrane interactions and biological activities of arapidly expnanding group of proteins (see, for example, Maltese (1990)FASEB J 4:3319; and Glomset et al. (1990) Trends Biochem Sci 15:139).Either farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoidscan be attached to specific proteins, with geranylgeranyl being thepredominant isoprenoid found on proteins (Fransworth et al. (1990)Science 247:320).

[0018] Three enzymes have been described that catalyze proteinprenylation: farnesyl-protein transferase (FPTase),geranylgeranyl-protein transferase type I (GGPTase-I), andgeranylgeranyl-protein transferase type-II (GGPTase-II, also called RabGGPTase). These enzymes are found in both yeast and mammalian cells(Schafer et al. (1992) Annu. Rev. Genet. 30:209-237). FPTase andGGPTase-I are α/β heterodimeric enzymes that share a common α subunit;the β subunits are distinct but share approximately 30% amino acidsimilarity (Brown et al. (1993). Nature 366:14-15; Zhang et al. (1994).J. Biol. Chem. 269:3175-3180). GGPTase II has different α and β subunitsand complexes with a third component (REP, Rab Escort Protein) thatpresents the protein substrate to the α/β catalytic subunits. Each ofthese enzymes selectively uses farnesyl diphosphate or geranylgeranyldiphosphate as the isoprenoid donor and selectively recognizes theprotein substrate. FPTase famesylates CaaX-containing proteins that endwith Ser, Met, Cys, Gln or Ala. GGPTase-I geranylgeranylatesCaaX-containing proteins that end with Leu or Phe. For FPTase andGGPTase-I, CaaX tetrapeptides comprise the minimum region required forinteraction of the protein substrate with the enzyme. GGPTase-IImodifies XXCC and XCXC proteins; the interaction between GGPTase-II andits protein substrates is more complex, requiting protein sequences inaddition to the C-terminal amino acids for recognition. Theenzymological characterization of these three enzymes has demonstratedthat it is possible to selectively inhibit one with little inhibitoryeffect on the others (Moores et al. (1991) J. Biol. Chem. 266:17438).

[0019] GGPTase I transfers the prenyl group from geranylgeranyldiphosphate to the sulphur atom in the Cys residue within the CAAXsequence. S Cerevisiae proteins such as the Ras superfamily proteinsRho1, Rho2, Rsr1/Bud1 and Cdc42 appear to be GGPTase substrates (Madauleet al. (1987) PNAS 84:779-783; Bender et al. (1989) PNAS 86:9976-9980;and Johnson et al. (1990) J Cell Biol 111:143-152).

[0020] III. Protein Kinase C

[0021] Members of the family of phospholipid-dependent,serine/threonine-specific protein kinases known collectively as proteinkinase C (PKC) respond to extracellular signals that act throughreceptor-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate todiacyl-glycerol (DAG) and inositol-1,4,5-trisphosphate (IP₃) (Hokin(1985) Annu. Rev. Biochem. 54, 205-235.). DAG serves as a secondmessenger to activate PKC (Takai et al. (1979) Biochem. Biophys. Res.Commun. 91, 1218-1224; Kishimoto et al. (1980) J. Biol. Chem. 255,2273-2276; Nishizuka 1986) Science 233, 305-312; and Nishizuka (1988)Nature 334, 661-665), and IP₃ functions to mobilize Ca²⁺ fromintracellular stores (Berridge et al. (1984) Nature 312, 215-321).Twelve distinct subtypes of mammalian PKC have been reported to date(Nishizuka, Y. (1992) Science 258, 607-614; Decker et al. (1994) TIBS19:73-77). The four initially identified isozymes, α, βI, βII, and γ,are structurally closely related to each other and display similarcatalytic properties.

[0022] Mammalian PKC is thought to play a pivotal role in the regulationof a host of cellular functions through its activation by growth factorsand other agonists. These functions include cell growth andproliferation, release of various hormones, and control of ionconductance channels. Indirect evidence suggests that PKC induces thetranscription of a wide array of genes, including the proto-oncogenesc-myc, c-fos, and c-sis, human collagenase, metallothionein II_(A), andthe SV40 early genes.

[0023] The PKC1 gene of budding yeast encodes a homolog of the α, β, andγ isoforms of mammalian Protein Kinase C that regulates aMAPK-activation pathway. Loss of PKC1 function results in a cell lysisdefect that is due to a deficiency in cell wall construction.

SUMMARY OF THE INVENTION

[0024] The present invention provides drug screening assays foridentifying pharmaceutically effective compounds that specificallyinhibit the biological activity of fungal GTPase proteins, particularlyGTPases involved in cell wall integrity, hyphael formation and othercell functions critical to pathogenesis. Briefly, as described ingreater detail below, Applicants have discovered the critical involvmentof Rho-like GTPase activities in cell wall integrity. For instance, thefungal Rho1 GTPase is required for glucan synthase activity, copurifieswith 1,3-β-glucan synthase, and is found to associate with the Gsc1/Fks1subunit of this complex in vivo. Rho1 is an regulatory subunit of1,3-β-glucan synthase, and accordingly this interaction, and theresulting enzyme complex, are potential therapeutic targets fordevelopment of antifungal agents. Moreover, Rho1 is required for proteinkinase C (PKC1) mediated MAPK activation, amd confers upon PKC1 theability to be stimulated by phosphatidylserine (PS), indicating thatRho1 controls signal transmission through PKC1. Loss of PCK1 activityresults in cell lysis. Also, we demonstrate that prenylation of Rho1 bya geranylgeranyl transferase is a critical step to maintenance of cellwall integrity in yeast. As described in the appended examples,prenylation of Rho1 is required for sufficient glucan synthase activity.Loss of Rho1 prenylation results in cell lysis. In general, a salientfeature of the subject assays is that the each is generated to detectagent which are potentially cytotoxic to a fungal cell, rather thanmerely cytostatic. Moreover, given the uniqueness of the therapeuticfungal targets of the present assays, e.g., relative to homolgousproteins in mammalian cells, the therapeutic targeting of Rho-likeGTPase(s) involvement in such interactions and complexes in yeastpresents an opportunity to define antifungal agents which are highlyselective for yeast cells relative to mammalian cells.

[0025] In one aspect, the present invention provides an assay foridentifying potential anti-fungal agents by targeting the GGPTase/GTPaseinteraction. For instance, the assay can be run by forming a reactionmixture including (i) a fungal geranylgeranyl transferase (GGPTase),(ii) a substrate for the GGPTase, such as a target polypeptidecomprising a fungal Rho-like GTPase such as Rho1, Rho2, Rsr1/Bud1 andCdc42, or a polypeptide portion thereof including at least one of (a) aprenylation site which can be enzymatically prenylated by the GGPTase,or (b) a GGPTase binding sequence which specifically binds the GGPTase,and (iii) a test compound. The interaction of the target polypeptidewith the GGPTase can be detected. A statistically significant decreasein the interaction of the target polypeptide and GGPTase in the presenceof the test compound, relative to the level of interaction in theabsence of the test compound (or other control), indicates a potentialanti-fungal activity for the test compound.

[0026] The reaction mixture can be a reconstituted protein mixture, acell lysate or a whole cell. For instance, the reaction mixture can be aprenylation system including an activated geranylgeranyl group, and thestep of detecting the interaction of the target polypeptide with theGGPTase includes detecting conjugation of the geranylgeranyl group tothe target polypeptide. In preferred embodiments of such prenylationsystems at least one of the geranylgeranyl group and the targetpolypeptide has a detectable label, and the level of geranylgeranylgroup conjugated to the target polypeptide is quantified by detectingthe label in at least one of the target polypeptide, free geranylgeranylgroups, and geranylgeranyl-conjugated target polypeptide. As illustratedbelow, the substrate target can incorporate a fluorescent (or other)label, the fluorescent characterization of which is altered by the levelof prenylation of the substrate target, e.g., the substrate target canbe a dansylated peptide substrate of the fungal GGPTase.

[0027] In other embodiments, the step of detecting the interaction ofthe target polypeptide with the GGPTase includes detecting the formationof protein-protein complexes including the target polypeptide with theGGPTase. For example, at least one of the GGPTase and the targetpolypeptide can include a detectable label, and the level ofGGPTase/target polypeptide complexes formed in the reaction mixture isquantified by detecting the label in at least one of the targetpolypeptide, the GGPTase, and GGPTase/target polypeptide complexes.Exemplary labels for such embodiments, and for the prenylation assaysabove, include radioisotopes, fluorescent compounds, enzymes, and enzymeco-factors. For instance, the detectable label can be a protein having ameasurable activity, and one of the PKC or GTPase is fusion proteinincluding the detectable label. In other exemplary embodiments,conjugation of the geranylgeranyl group to the target polypeptide isdetected by an immunoassay.

[0028] Where the reaction mixture is a whole cell, the cell willpreferably include heterologous nucleic acid recombinantly expressingone or more of the fungal GGPTase subunits and target polypeptide. Incertain preferred embodiments, the cell will also include a heterologousreporter gene construct having a reporter gene in operable linkage witha transcriptional regulatory sequence sensitive to intracellular signalstransduced by interaction of the target polypeptide and GGPTase.

[0029] In one preferred embodiment, the assay includes forming acell-free reaction mixture including: (i) a fungal GGPTase, (ii) aGGPTase substrate, e.g., a target polypeptide comprising a fungalRho-like GTPase, or a polypeptide portion thereof including aprenylation site, (iii) an activated geranylgeranyl group, (iv) adivalent cation, and (v) a test compound. The assay is derived to detectconjugation of the gernaylgernayl group of the target polypeptide in thereaction mixture, and a statistically significant decrease in theprenylation of the target polypeptide and GGPTase in the presence of thetest compound, relative to an appropriate control, indicates a potentialanti-fungal activity for the test compound.

[0030] In another preferred embodiment, the method utilizes aninteraction trap system including (a) a first fusion protein comprisingat least a portion of a fungal GGPTase subunit, (b) a second fusionprotein comprising at least a portion of a fungal GTPase, and (c) areporter gene, including a transcriptional regulatory sequence sensitiveto interactions between the GGPTase portion of the first fusion proteinand the GTPase portion of the second polypeptide. After contacting theinteraction trap system with a candidate agentthe level of expression ofa reporter gene is measured and compared to the level of expression inthe absence of the candidate agent. A decrease in the level ofexpression of the reporter gene in the presence of the candidate agentis indicative of an agent that inhibits interaction of the GGPTase andGTPase.

[0031] In still another embodiment, the assay is derived from a arecombinant cell expressing a recombinant form of one or more of afungal GGPTase and a fungal Rho-like GTPase. The cell is contacted witha test compound, and the level of interaction of the GGPTase andRho-like GTPase is detected. A statistically significant change in thelevel of interaction of the GGPTase and Rho-like GTPase is indicative ofan agent that modulates the interaction of those two proteins. Inpreferred embodiments, one or both of a GGPTase subunit or the Rho-likeGTPase are fusion proteins, e.g., the fusion protein providing adetectable label and/or an affinity tag for purification. In a preferredembodiment, the Rho-like GTPase is a fusion protein further comprising atranscriptional regulatory protein, and level of prenylation of theRho-like GTPase is detected by measuring the level of expression of areporter gene construct which is sensitive to the transcriptionalregulatory protein portion of the fusion protein, wherein inhibition ofprenylation of the fusion protein results in loss of membranepartitioning of the fusion protein and increases expression of thereporter gene construct.

[0032] In other preferred embodiments, the level of interaction of theGGPTase and Rho-like GTPase is detected by detecting prenylation of theRho-like GTPase.

[0033] In yet another preferred embodiment, the assay is generated froma set of cells in which prenylation of endogenous Rho-like GTPases byGGPTase I is made dispensible. According to this embodiment, the assayprovides a first test cell in which one or more Rho-like GPTases aremutated to be a substrate for a farnesyl transferase expressed by thecell such that GGPTase I is dispensible for cell growth; and a secondtest cell identical to the first cell except that the Rho-like GTPasesare substrates for GGPTase I and are indispensible for cell growth. Thefirst and second cells are contacted with a candidate agent, and thelevel of prenylation of the Rho-like GTPases in first and second testcells are compared. A statistically significant decrease in theprenylation of the GTPases in the second test cell, relative to thelevel of prenylation of the GTPase in the first cell, is indicative ofan agent that inhibits interaction of a GGPTase and GTPase.

[0034] Yet another aspect of the present invention, the subject assaysare derived for detecting agents which disrupt the formation of, orfunction of fungal protein complexes including Rho-like GTPases and PKCproteins. In one embodiment, the assay provides a reaction mixtureincluding a fungal Rho-like GTPase, a fungal protein kinase C (PKC), anda test compound. Interaction of the Rho-like GTPase and PKC is detectedin the reaction mixture, wherein a statistically significant decrease inthe interaction of the Rho-like GTPase and PKC in the presence of thetest compound, relative to the level of interaction in the absence ofthe test compound, indicates a potential antifungal activity for thetest compound.

[0035] The reaction mixture can be a reconstituted protein mixture, acell lysate or a whole cell. In preferred embodiments, the reactionmixture is a kinase system including ATP and a PKC substrate, and thestep of detecting interaction of the GTPase and PKC includes detectingphosphorylation of the PKC substrate by a PKC/GTPase complex.Preferably, at least one of the PKC substrate and ATP includes adetectable label, and the level of phosphorylation of the PKC substrateis quantified by detecting the label in at least one of thephosphorylated PKC substrate or ATP. For instance, the PKC substrate mayinclude a fluorescent (or other) label, the fluorescent characterizationof which is altered by the level of phosphorylation of the PKCsubstrate.

[0036] In other preferred embodiments, the step of detecting theinteraction of the GTPase with the PKC includes detecting the formationof protein-protein complexes including the GTPase and PKC. For instance,at least one of the PKC and GTPase includes a detectable label, and thelevel of PKC/GTPase complexes formed in the reaction mixture isquantified by detecting the label in at least one of the GTPase, thePKC, and PKC/GTPase complexes. For instance, phosphorylation of the PKCsubstrate is detected by immunoassay.

[0037] Cell-based assays are also provided, including cells comprisingreporter gene constructs sensitive to PKC/GTPase complexes. In oneembodiment, PKC/GTPases interaction trap assays are used for drugscreening according to the present invention.

[0038] In still another aspect of the present invention, the subjectassays are derived for detecting agents which disrupt the formation of,or function of fungal protein complexes including Rho-like GTPases andglucan synthase complexes or subunits thereof. In a preferredembodiment, the assay includes forming a reaction mixture including afungal Rho-like GTPase, a fungal glucan synthase complex or subunitthereof (collectively “GS protein”), and a test compound. Theinteraction of the Rho-like GTPase and GS protein can be detected in thereaction mixture. Similar to the assay embodiments set out above, astatistically significant decrease in the interaction of the Rho-likeGTPase and GS protein in the presence of the test compound, relative tothe level of interaction in the absence of the test compound, indicatesa potential antifungal activity for the test compound.

[0039] The reaction mixture can be a reconstituted protein mixture, acell lysate or a whole cell. In preferred embodiments, the reactionmixture is a glucan synthesis system including a GTP and a UDP-glucose,and the step of detecting interaction of the GTPase and GS proteinincludes detecting formation of glucan polymers in the reaction mixture,e.g., the UDP-glucose can include a detectable label, and the level ofglucan polymer formation is quantified by detecting the labeled glucanpolymers.

[0040] In other embodiments, the step of detecting the interaction ofthe GTPase with the GS protein includes detecting the formation ofprotein-protein complexes including the GTPase and GS protein. As above,at least one of the GS protein and GTPase can include a detectablelabel, and the level of GS protein/GTPase complexes formed in thereaction mixture is quantified by detecting the label in at least one ofthe GTPase, the GS protein, and GS protein/GTPase complexes.Alternatively, the formation of protein-protein complexes including theGTPase and GS protein is detected by an immunoassay.

[0041] As above, cell-based assays are also provided, including cellscomprising reporter gene constructs sensitive to GS/GTPase complexes.Permeabilization of cells due to disruption of GS activity by the testcompound can also be detected by loss of cytoplasmic localization orcytoplasmic exclusion (depending on the embodiment) of a detectablelabel.

[0042] For each of the assay embodiments set out above, the assay ispreferably repeated for a variegated library of at least 100 differenttest compounds, though preferably libraries of at least 10³, 10 ⁵, 10 ⁷,and 10⁹ compunds are tested. The test compound can be, for example,small organic molecules, and/or natural product extracts.

[0043] Also, in preferred embodiments of the subject assay, one or moreof the GTPase of other proteins which interacting with the GTPase (e.g.,GGPTase subunits, PKC and glucan synthase subunits) are derived from ahuman pathogen which is implicated in mycotic infection.

[0044] The subject assay also preferably includes a further step ofpreparing a pharmaceutical preparation of one or more compoundsidentified as having potential antifungal activity.

[0045] Still another aspect of the invention concerns variouscompositions and reagents for performing the subject drug screeningassays. For instance, the present invention provides a variety ofrecombinant cells expressing one or more different fungal proteinsimplicated as targets in the subject screening assays. In a preferredembodiment, the recombinant cell includes exogenous nucleic acid (e.g.,expression vectors) encoding a fungal Rho-like GTPase. In a morepreferred embodiment, the recombinant cell includes (i) exogenousnucleic acid(s) encoding one or more subunits of a fungal geranylgeranylprotein transferase (GGPTase), and (ii) exogenous nucleic acid encodinga fungal Rho-like GTPase or a fragment thereof including at least one of(a) a prenylation site which can be enzymatically prenylated by theGGPTase, or (b) a GGPTase binding sequence which specifically binds theGGPTase. In still other preferred embodiments, the cell inlcudes (i)exogenous nucleic acid encoding a fungal Rho-like GTPase, and (ii)exogenous nucleic acid encoding a fungal protein selected from the groupconsisting of a fungal protein kinase C (PKC) or one or more subunits ofa fungal glucan synthase.

[0046] The nucleic acids encoding the GGPTase, GTPase, PKC and/or glucansynthase are preferably derived from a human pathogen which isimplicated in mycotic infection. For instance, the recombinant genes canbe derived from fungus involved in such mycotic infections as selectedfrom a group consisting of candidiasis, aspergillosis, mucormycosis,blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis,penicilliosis, conidiosporosis, nocaidiosis, coccidioidomycosis,histoplasmosis, maduromycosis, rhinosporidosis, monoliasis,para-actinomycosis, and sporotrichosis. To further illustrate, theexpression vectors can be generated from genes cloned from humanpathogen selected from a group consisting of Candida albicans, Candidastellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei,Candida pseudotropicalis, Candida quillermondii, Candida rugosa,Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger,Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopusoryzae, Absidia corymbifera, Absidia ramosa, and Mucor pusillus. Anothersource for recombinant genes is the human pathogen is Pneumocystiscarinii.

[0047] In preferred embodiments, the cell is a recombinantly manipulatedyeast cell selected from the group consisting of such genuses asKluyverei, Schizosaccharomyces, Ustilaqo and Saccharomyces, though aprefered host cell is the Schizosaccharomyces cerivisae cell. Moreover,the host cell can be constitutively or inducibly defective for anendogenous activity corresponding to one or more of the GGPTase andGTPase encoded by the exogenous nucleic acids.

[0048] In similar fashion, another aspect of the present inventionconcerns reconstituted protein mixtures or cell lysate mixturesincluding a recombinant fungal Rho-like GTPase, .e.g, or a fragmentthereof including at least one of (a) a prenylation site which can beenzymatically prenylated by the GGPTase, or (b) a GGPTase bindingsequence which specifically binds the GGPTase, along with one or more ofa recombinant fungal glucan synthase, a recombinant fungal GGPTase,and/or a recombinant fungal PKC. As above, the fungal target proteinsare preferably derived from a human pathogen which is implicated inmycotic infection.

[0049] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

BRIEF DESCRIPTION OF THE DRAWINGS

[0050]FIG. 1. Overexpression of PKC1 suppresses the cell lysis defect ofa rho1^(ts) mutant. (A) The rho1-5 allele lyses at restrictivetemperature. Yeast strains patched on a YPD plate were incubated at 23°C. for 3 days, then shifted overnight to 37° C. The patches were assayedin situ for release of alkaline phosphatase as an indication of celllysis. 1, wild-type; 2, rho1-3; 3, rho1-5; 4, pkc1^(ts) (stt1-1;SYT11-12A). (B) An episomal plasmid (YEp352) with or without PKC1 wastransformed into the rho1^(ts) mutants (rho1-3 and rho1-5).Transformants were streaked onto a YPD plate and incubated at 37° C. for3 days.

[0051]FIG. 2. RHO1 is required for Mpk1 activation in response to heatshock. (Top panel) Phosphorylation of myelin basic protein (MBP) byMpk1^(HA) immunoprecipitated from extracts of cells shifted from growthat 23° C. to 39° C. for 30 min. This treatment did not affect theviability of the mutant strains (data not shown). Mpk1 activity inrho1-5 (lanes 4 and 5) and rho1-3 (lanes 6 and 7) relative to wild-type(RHO1; lane 1-3) maintained at 23° C. (lane 1) is indicated. (Bottompanel) Immunoblot of immunoprecipitated Mpk1^(HA).

[0052]FIG. 3. PKC1 associates with Rho1 in vivo and in vitro. (A)^(HA)Rho1 was immunoprecipitated from extracts of cells growing at 23°C. (lane 4), or shifted from 23° C. to 39° C. for 30 min (lane 6).^(HA)Rho1 immunoprecipitates (left) and whole-cell extracts (100 μgprotein; right) were analyzed by immunoblot with anti-PKC1 antibodies(top panels), or with anti-HA (to detect ^(HA)Rho1; bottom panels).Untagged Rho1 was used as a negative control (lanes 1, 2, and 7). Bandindicated by * is derived from immunoprecipitating antibodies. (B)Recombinant GST-Rho1 (1 μg), purified from Sf9 insect cells and bound toglutathione agarose beads, was preloaded with the indicated guaninenucleotide (lanes 2-5). Soluble yeast cell extract (400 μg protein)containing PKC1^(HA) was incubated with the beads (lanes 1, 3, and 5),and bound PKC1^(HA) was detected by immunoblot analysis. A control inwhich naked glutathione agarose beads were used (lane 1) demonstratesdependence of PCK1^(HA) binding on GST-Rho1.

[0053]FIG. 4. Rho1 allows cofactors to activate PKC1. (A)Phosphorylation of synthetic Bck1 peptide by PKC1^(HA)immunoprecipitated from 50 μg of soluble yeast cell extract protein.Recombinant GST-Rho1 or GST-Cdc42 (1 μg) was preloaded with theindicated guanine nucleotide. Cofactors (80 μg/ml PS, 8 μg/ml DAG, and100 μM CaCl₂) were added to the reaction where indicated. Lanes 1 and 2are control reactions with no GTPase. Mean and standard error for threeexperiments is shown. (B) PS alone is sufficient to stimulate PKC1 fullyin the presence of Rho1. Phosphorylation of Bck1 peptide by PKC1^(HA) inthe presence of GTPγS-bound GST-Rho1 and the indicated cofactors.Conditions were as in A, except for PMA (16 ng/ml). Concentrations of PSas low as 8 μg/ml fully activated PKC1 (data not shown).

[0054]FIG. 5. Model for the dual role of Rho1 in the maintenance of cellintegrity.

[0055]FIG. 6. GS activity from rho1 mutants (See reference of Example3). (A) GS activity is thermolabile in rho1^(ts) mutants. Crude extractswere made from cells growing at room temperature, and assayed for GSactivity at the indicated temperatures in the presence of 50 μM GTPγS.(B) Reconstitution of GS activity in rho1-3 membranes with recombinantRho1. GS activity in rho1-3 membrane fractions was measured at 37° C. inthe presence of 1 μg of the indicated recombinant GTPase and 50 μM GTPγS(19). (C) Reconstituted GS activity requires GTP. GS activity inwild-type membranes or rho1-3 membranes complemented with 1 μg ofGST-Rho1 was measured at 37° C. in the presence of the indicated guaninenucleotide (20 μM). Results are expressed as percent activity relativeto GTPγS.

[0056]FIG. 7. GS activity in a constitutively active RHO1 mutant is GTPindependent. Cultures of rho1-3 cells harboring plasmids with eitherRHO1 or RHO1-Q68H (M. S. Boguski et al. (1992) New Biol. 4:408) underthe control of the inducible GAL1 promoter were grown at roomtemperature in medium containing 2% raffinose (repressing conditions).Galactose was added (to 2%) to half of each culture, and cells werecultured for an additional 4 h to induce expression of RHO1. GS activityin membrane fractions was assayed at 37° C. in the presence or absenceof GTPγS.

[0057]FIG. 8. Rho1 and Gsc1/Fks1 are enriched during purification of GS.GS was purified from a wild-type strain (A451; 3). (A) Immunoblotanalysis of Rho1 (upper) and Gsc1/Fks1 (lower) through purification (Seereference 20 of Example 2). (B) GS specific activity throughpurification. Purification steps were: lane 1, membrane fraction; lane2, detergent extract; lane 3, first product entrapment; lane 4, secondproduct entrapment.

[0058]FIG. 9. (A) Coimmunoprecipitation of Rho1 with Gsc1/Fks1 (21).Partially purified GS was incubated with anti-Gsc1/Fks1 monoclonalantibodies, 1A6 (lane 1) and 1F4 (lane 2), and anti-human endothelin Btype receptor (lane 3) (3). Immunoprecipitates were analyzed by SDS-PAGEfollowed by immunoblotting. (B) Colocalization of Gsc1/Fks1 and Rho1 atsites of cell wall remodeling (See reference 22 of Example 2). Indirectimmunofluorescence microscopy was used to visualize Gsc1/Fks1 and^(HA)Rho1 in double-stained cells.

[0059]FIG. 10. Alignments of the β-subunits of GGPTase-Is showingcal1/cdc43 mutations. Positions of the cal1/cdc43 mutations are shownunder the box representing the CAL1/CDC43 coding region. The closed boxrepresents the homologous region among the β-subunits of the proteinisoprenyltransferase. Cluster was used to align Cal1p, and theβ-subunits of the S. pombe, rat and human GGPTase-Is near the cal1/cdc43mutation points.

[0060]FIG. 11. Overproduction of CDC42 is toxic in cal1-1 cells. cal1-1(1), cdc43-2 (2), cdc43-3 (3) cdc43-4 (4) cdc43-5 (5), cdc43-6 (6),cdc43-7 (7) and wild-type strain (M. S. Boguski et al. (1992) New Biol.4:408) harboring pGAL-CDC42 were streaked on the plate containingglucose (A) or galactose (B), and incubated at 23° C. for 1 week.

[0061]FIG. 12. Fractionation of Rho1p and Cdc42p in wild-type and mutantstrains. Yeast strains were grown to midlog phase at the permissivetemperature (23° C.), shifted to the restrictive temperature, collectedafter 2 hr (37° C.), and the cell lysates were prepared. Rho1p wasdetected by Western blotting analysis with guinea pig polyclonalantibody against Rho1p. In order to express HA-tagged version of Cdc42p,yeast strains transformed with pYO920 were incubated at 23° C. in 2%galactose-containing medium for 6 hr before the temperature shift.HA-tagged version of Cdc42p was detected by Western blotting analysiswith 12CA5. WT, YPH500; cal1-1, YOT159-3C; cdc43-5, YOT435-1A.

[0062]FIG. 13. Reduced GS activity in the membrane fractions of GGPTaseI-deficient cells. Cultures of wild-type (YPH500), cal1-1 (YOT159-3C),cdc43-5 (YOT435-1A) cells were grown at room temperature in YPD medium.GS activity in membrane fractions was assayed at 30° C. according toInoue et al. (1995) Eur. J. Biochem. 231: 845. Reconstitution of GSactivity in cal1-1 membrane was performed with recombinant mutant Rho1(G19V) which is constitutively active for its activity.

[0063]FIG. 14. Thin section electron micrograph of Pkc1-depleted cellsdemonstrating cell lysis.

DETAILED DESCRIPTION OF THE INVENTION

[0064] The use of, and need for anti-fungal agents is widespread andranges from the treatment of mycotic infections in animals; to additivesin feed for livestock to promote weight gain; to disinfectantformulations. In general, a salient feature of effective anti-fungalagents is that the agent is cytotoxic to a fungal cell rather than onlycytostatic. The mere knowledge that a particular protein is critical tocell growth is accordingly not sufficient to render that protein asuitable target for generation of anti-fungal agents. Rather, assayswhich are useful for identifying potential anti-fungal agents shouldtarget a fungal bioactivity which, when altered in a particular manner,results in cell death rather than quiescence or sporulation. Forexample, as is illustrated in FIG. 14, cell lysis is a preferred outcometo treatment with the potential antifungal agent in order to ensuredestruction of the pathogen. Moreover, at least for anti-fungal agentswhich are to be administered to humans and other animals, thetherapeutic index is preferably such that toxicity to the host isseveral orders of magnitude less than it is for the targeted fungus.

[0065] The present invention relates to rapid, reliable and effectiveassays for screening and identifying pharmaceutically effectivecompounds that specifically inhibit the biological activity of fungalGTPase proteins, particularly GTPases involved in cell wall integrity,hyphael formation, and other cellular functions critical topathogenesis.

[0066] The cell wall of many fungus, as set out above, is required tomaintain cell shape and integrity. The main structural componentresponsible for the rigidity of the yeast cell wall is 1,3-β-linkedglucan polymers with some branches through 1,6-β-linkages. Thebiochemistry of the yeast enzyme catalyzing the synthesis of1,3-β-glucan chains has been studied extensively, but little waspreviously known at the molecular level about the genes encodingsubunits of this enzyme. Only a pair of closely related proteins(Gsc1/Fks1 and Gsc2/Fks2) had previously been described as subunits ofthe 1,3-β-glucan synthase (GS) (Inoue et al., (1995) Eur. J. Biochem.231:845; and Douglas et al., (1994) PNAS 91:12907). GS activity in manyfungal species, including S. cerevisiae, requires GTP or anon-hydrolyzable analog (e.g. GTPγS) as a cofactor, suggesting that aGTP-binding protein stimulates this enzyme (Mol et al. (1994) J. Biol.Chem. 269:31267).

[0067] As described in the appended examples, we demonstrate that theRho1 GTPase activity is required for glucan synthase activity,copurifies with 1,3-β-glucan synthase, and is found to associate withthe Gsc1/Fks1 subunit of this complex in vivo. Both proteins were alsofound to reside predominantly at sites of cell wall remodeling.Therefore, Rho1 is an regulatory subunit of 1,3-β-glucan synthase, andaccordingly this interaction, and the resulting enzyme complex, arepotential therapeutic targets for development of antifungal agents.Moreover, given the uniqueness of the yeast glucan cell wall relative tomammalian cells, the therapeutic targeting of Rho-like GTPase(s)involvement in glucan synthase complexes in yeast presents anopportunity to define antifungal agents which are highly selective foryeast cells relative to mammalian cells.

[0068] We have also discovered other interactions with Rho1-like GTPasewhich are consequential to cell integrity in yeast. As described in theappended examples, we find that Rho1 is required for protein kinase C(PKC1) mediated MAPK activation. Moreover, PKC1 co-immunoprecipitateswith Rho1 in yeast extracts, and recombinant Rho1 associates with PKC1in vitro in a GTP-dependent manner. Moreover, the data provided hereindemonstrates that recombinant Rho1 confers upon PKC1 the ability to bestimulated by phosphatidylserine (PS), indicating that Rho1 controlssignal transmission through PKC1. This applications provides the firstexample of a PKC isoform whose stimulation by cofactors is dependent ona GTPase, and provides the basis for yet other drug screening assaysthat target the interaction of a PKC and GTPase, or the catalyticactivity of the resulting complex. Furthermore, no mammalian PKCactivities have been reported to require a G-protein co-factor,suggesting that the fungal Rho/PKC complex represents a specific targetfor developing antiproliferative agents selective for yeast cells.

[0069] Finally, we have demonstrated that prenylation of Rho1 by ageranylgeranyl transferase is a critical step to maintenance of cellwall integrity in yeast. As described in the appended examples,prenylation of Rho1 is required for sufficient glucan synthase activity.Taken together with the results respecting Rho1's participation as a GSsubunit, we demonstrate that not only is the prenylatin of Rho1 byGGPTase I critical to cell growth, but inhibition of the prenylationreaction is a potential target for developing a cytotoxic agent forkilling various fungi. Moreover, the relatively high divergence betweenfungal and human GGPTase subunits suggests that selectivity for thefungal GGPTase activity may be obtained to provide antifungal agentshaving desirable therapeutic indices.

[0070] In one embodiment, the subject assay comprises a prenylationreaction system that includes a fungal geranylgeranyl proteintransferase (GGPTase), a fungal GTPase protein, or a portion thereof,which serves as a prenylation target substrate, and an activatedgeranylgeranyl moiety which can be covalent attached to the prenylationsubstrate by the GGPTase. The level of prenylation of the targetsubstrate brought about by the system is measured in the presence andabsence of a candidate agent, and a statistically significant decreasein the level prenylation is indicative of a potential anti-fungalactivity for the candidate agent.

[0071] As described below, the level of prenylation of the GTPase targetprotein can be measured by determining the actual concentration ofsubstrate:geranylgeranyl conjugates formed; or inferred by detectingsome other quality of the target substrate affected by prenylation,including membrane localization of the target. In certain embodiments,the present assay comprises an in vivo prenylation system, such as acell able to conduct the target substrate through at least a portion ofa geranylgeranyl conjugation pathway. In other embodiments, the presentassay comprises an in vitro prenylation system in which at least theability to transfer isoprenoids to the GTPase target protein isconstituted. Still other embodiments provide assay format which detectprotein-protein interaction between the GGPTase and a target protein,rather than enzymatic activity per se.

[0072] With respect to the interaction of the fungal GTPase with othercellular components, and the significance of those interactions to cellwall integrity, another aspect of the present invention relates toassays which seek to identify agents which alter protein-proteininteractions involving a fungal GTPase and PKC or glucan synthasesubunits, or which inhibit the catalytic activity of a protein complexresulting from such interactions. For instance, as described in moredetail below, one therapeutic target of interest are glucan synthasecomplexes which include a Rho1-like GTPase. In another embodiment, thetherapeutic target is a protein kinase C complex including a GTPase. Theparticular assay format selected will reflect the desire to identifycompounds which disrupt protein-protein interactions and thereby alterthe enzyme complex, or which disrupt the interaction with, and chemicalalteration of a given substrate by the enzyme complex. For instance, theinteraction with, and chemical alteration of a given substrate by theenzyme complex. For instance, the interaction of Rho1 with the glucansynthase subunit Gce1 can be the screening target in some embodiments,while the synthase activity of the resulting complex can be thescreening target in other embodiments. Likewise, screening assaystargeting PKC1/Rho1 complex can provide agents which disrupt theformation of the complex, or target the complex's interaction withsubstrate proteins.

[0073] As described herein, inhibitors of a fungal GTPase bioactivityrefer generally to those agents which may act anywhere along theprenylation pathway, e.g., from the reaction steps leading up to andincluding conjugation of an isoprenoid to the GTPase target, to theinteraction of the GTPase protein with other cellular proteins, such asglucan synthase subunits and/or PKC. A subset of this class ofinhibitors comprises the prenylation inhibitors, which include thoseagents that act at the level of preventing conjugation of geranylgeranylmoieties to the target GTPase, rather than at the steps ofprotein-protein interactions involving the prenylated GTPase, e.g., aspart of enzymatic complexes. Moreover, as will be clear from thefollowing description, particular embodiments of the present assay canbe chosen so as to discriminate between prenylation inhibitors andinhibitors of prenylated-GTPase complexes.

[0074] For convenience, certain terms employed in the specification,examples, and appended claims are collected here.

[0075] As used herein, “recombinant cells” include any cells that havebeen modified by the introduction of heterologous DNA. Control cellsinclude cells that are substantially identical to the recombinant cells,but do not express one or more of the proteins encoded by theheterologous DNA, e.g., do not include or express a recombinantRho1-like GTPase, a recombinant GGPTase, a recombinant glucan synthaseand/or a recombinant PKC1.

[0076] The terms “recombinant protein”, “heterologous protein” and“exogenous protein” are used interchangeably throughout thespecification and refer to a polypeptide which is produced byrecombinant DNA techniques, wherein generally, DNA encoding thepolypeptide is inserted into a suitable expression vector which is inturn used to transform a host cell to produce the heterologous protein.That is, the polypeptide is expressed from a heterologous nucleic acid.

[0077] As used herein, “heterologous DNA” or “heterologous nucleic acid”include DNA that does not occur naturally as part of the genome in whichit is present or which is found in a location or locations in the genomethat differs from that in which it occurs in nature. Heterologous DNA isnot endogenous to the cell into which it is introduced, but has beenobtained from another cell. Generally, although not necessarily, suchDNA encodes RNA and proteins that are not normally produced by the cellin which it is expressed. Heterologous DNA may also be referred to asforeign DNA. Any DNA that one of skill in the art would recognize orconsider as heterologous or foreign to the cell in which is expressed isherein encompassed by heterologous DNA. Examples of heterologous DNAinclude, but are not limited to, isolated DNA that encodes a Rho1-likeGTPase, a GGPTase, a glucan synthase and/or a PKC1.

[0078] “Inactivation”, with respect to genes of the host cell, meansthat production of a functional gene product is prevented or inhibited.Inactivation may be achieved by deletion of the gene, mutation of thepromoter so that expression does not occur, or mutation of the codingsequence so that the gene product is inactive (constitutively orinducibly). Inactivation may be partial or total.

[0079] “Complementation”, with respect to genes of the host cell, meansthat at least partial function of inactivated gene of the host cell issupplied by an exogenous nucleic acid. For instance, yeast cells can be“mammalianized”, and even “humanized”, by complementation of Rho1 withmammalian homologs such as RhoA.

[0080] As used herein, a “reporter gene construct” is a nucleic acidthat includes a “reporter gene” operatively linked to a transcriptionalregulatory sequences. Transcription of the reporter gene is controlledby these sequences. The transcriptional regulatory sequences include thepromoter and other regulatory regions, such as enhancer sequences, thatmodulate the activity of the promoter, or regulatory sequences thatmodulate the activity or efficiency of the RNA polymerase thatrecognizes the promoter, or regulatory sequences are recognized byeffector molecules.

[0081] The term “substantially homologous”, when used in connection withamino acid sequences, refers to sequences which are substantiallyidentical to or similar in sequence, giving rise to a homology inconformation and thus to similar biological activity. The term is notintended to imply a common evolution of the sequences.

[0082] The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein.

[0083] As used here, the terms “geranylgeranyl protein transferase” and“GGPTase are art recognized and refer to the enzyme complexesresponsible for the covalent modification of proteins withgeranylgeranyl moieties. Particular reference to fungal GGPTasessub-types such as GGPTase-I, or the subunits of a fungal GGPTase, suchas cdc43 and RAM2 (unless otherwise evident from the contest) isintended to refer generically to the analogous GGPTase complex and/orsubunits in any fungal cell. Accordingly, reference to the subunit cdc43(also referred to as CAL1 and DPR1) refers to the S. cerevisiae-isubunit as well as homologous proteins in that cell or other fungi whichform a GGPTase I enzyme complex.

[0084] Likewise, the terms “Rho-like GTPase” and “fungal GTPase” willrefer generally to GTPases related structurally to the yeast GTPasesRho1, Rho2, cdc42, and/or Rsr1/Bud1, whether the enzyme is isolated fromS. cerevisiae or other fungi.

[0085] In similar fashion, the term “glucan synthase” refers genericallyto fungal enzymes involved in synthesis of a β-(1,3)-glucan andcomprised of subunits including Gsc1 (also called Fks1) homologs andRho-like GTPases. As above, reference to a “Gsc1 subunit” refers to theS. cerevisiae-i protein as well as structurally and functionally relatedhomologs from other fungi.

[0086] The terms “PKC” and “PKC1” are also used generically to refer toprotein kinase C homologs in fungi, and other fungal homologs of thePKC1 protein of S. cerevisiae, respectively.

[0087] The terms “fungi” and “yeast” are used interchangeably herein andrefer to the art recognized group of eukaryotic protists known as fungi.That is, unless clear from the context, “yeast” as used herein canencompass the two basic morphologic forms of yeast and mold anddimorphisms thereof.

[0088] The present invention provides a systematic and practicalapproach for the identification of candidate agents able to inhibit oneor more of the cellular functions of fungal GTPase proteins. In ageneral sense, the assays of the present invention evaluate the abilityof a compound to modulate binding between a GTPase protein and anotherprotein, whether the GTPase is acting as a subunit of a multiproteincomplex or as a substrate for modification. The assays may be formattedto evaluate the ability of a compound to modulate (i) protein complexeswhich include a GTPase protein; (ii) the enzymatic activity of suchmultiprotein complexes; or (iii) the enzymatic activity which produces aprenylated GTPase.

[0089] Exemplary compounds which can be screened for activity againstfungal GTPase activity include peptides, nucleic acids, carbohydrates,small organic molecules, and natural product extract libraries, such asisolated from animals, plants, fungus and/or microbes.

[0090] Cell-free Assay Formats

[0091] In many drug screening programs which test libraries of compoundsand natural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins or cell-lysates, are oftenpreferred as “primary” screens in that they can be generated to permitrapid development and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with upstream ordownstream elements. Accordingly, in an exemplary screening assay of thepresent invention, a reaction mixture is generated to include a fungalGTPase polypeptide, compound(s) of interest, and a “target polypeptide”,e.g., a protein, which interacts with the GTPase polypeptide, whether asa prenylating activity, or by some other protein-protein interaction.Exemplary target polypeptides include GGPTase activities such as GGPTaseI, PKC homologs such as PKC1, and glucan synthase subunits such as Gsc1.Detection and quantification of the enzymatic conversion of the fungalGTPase, or the formation of complexes containing the fungal GTPaseprotein, provide a means for determining a compound's efficacy atinhibiting (or potentiating) complex the bioactivity of the GTPase. Theefficacy of the compound can be assessed by generating dose responsecurves from data obtained using various concentrations of the testcompound. Moreover, a control assay can also be performed to provide abaseline for comparison.

[0092] In one embodiment, the subject drug screening assay comprises aprenylation system, e.g. a reaction mixture which enzymaticallyconjugates isoprenoids to a target protein, which is arranged to detectinhibitors of the prenylation of a Rho-like GTPase with a geranylgeranylgroup. For instance, in one embodiment of a cell-free prenylationsystem, one or more cell lysates including a fungal GGPTase, a fungalRho-like GTPase (or substrate analog thereof), and an activatedgeranylgeranyl group are incubated with the test compound and the levelof prenylation of the Rho-like GTPase substrate is detected. Lysates canbe derived from cells expressing one or more of the relevant proteins,and mixed appropriately (or spilled) where no single lysate contains allthe components necessary for generating the prenylation system. Inpreferred embodiments, one or more of the components, especially thesubstrate target, are recombinantly produced in a cell used to generatea lysate, or added by spiking a lysate mixture with a purified orsemi-purified preparation of the substrate. These embodiments haveseveral advantages including: the ability to use a labeled substrate,e.g. a dansylated peptide, or fusion protein for facilitatingpurification e.g. a Rho1-GST fusion protein; the ability to carefullycontrol reaction conditions with respect to concentrations of reactants;and where targets are derived from fungal pathogens, the ability to workin a non-pathogenic system by recombinantly or synthetically producingby components from the pathogen for constituting the prenylation system.

[0093] The prenylates can be derived from any number of cell types,ranging from bacterial cells to yeast cells to cells from metazoanorganisms including insects and mammalian cells. To illustrate, a fungalprenylation system can be reconstituted by mixing cell lysates derivedfrom insect cells expressing fungal GGPTase subunits cloned intobaculoviral expression vectors. For example, the exemplary GGPTase-Iexpression vectors described below in the section Reagents-i can berecloned into baculoviral vectors (e.g. pVL vectors), and recombinantGGPTase-I produced in transfected spodoptera-i fungiperda cells. Thecells can than be lysed, and if the RAM2 and CDC43 subunits are producedby different sets of cells, cell lysates can be accordingly mixed toproduce an active fungal GGPTase. The level of activity can be assessedby enzymatic activity, or by quantitating the level of expression bydetecting, e.g., an exogenous tag added to the recombinant protein.Substrate and activated geranylgeranyl diphosphate can be added to thelysate mixtures. As appropriate, the transfected cells can be cellswhich lack an endogenous GGTase activity, or the substrate can be chosento be particularly sensitive to prenylation by the exogenous fungalGGTPase relative to any endogenous activity of the cells. In othercell-free embodiments of the present assay, the prenylation systemcomprises a reconstituted protein mixture of at least semi-purifiedproteins. By semi-purified, it is meant that the proteins utilized inthe reconstituted mixture have been previously separated from othercellular proteins. For instance, in contrast to cell lysates, theproteins involved in conjugation of geranylgeranyl moieties to a targetprotein, together with the target protein, are present in the mixture toat least 50% purity relative to all other proteins in the mixture, andmore preferably are present at 90-95% purity. In certain embodiments ofthe subject method, the reconstituted protein mixture is derived bymixing highly purified proteins such that the reconstituted mixturesubstantially lacks other proteins which might interfere with orotherwise alter the ability to measure specific prenylation rates of thetarget GTPase substrate.

[0094] Each of the protein components utilized to generate thereconstituted prenylation system are preferably isolated from, orotherwise substantially free of, other proteins normally associated withthe proteins in a cell or cell lysate. The term “substantially free ofother cellular proteins” (also referred to herein as “contaminatingproteins”) is defined as encompassing individual preparations of each ofthe component proteins comprising less than 20% (by dry weight)contaminating protein, and preferably comprises less than 5%contaminating protein. Functional forms of each of the componentproteins can be prepared as purified preparations by using a cloned geneas described below and known in the art. By “purified”, it is meant,when referring to the component protein preparations used to generatethe reconstituted protein mixture, that the indicated molecule ispresent in the substantial absence of other biological macromolecules,such as other proteins (particularly other proteins which maysubstantially mask, diminish, confuse or alter the characteristics ofthe component proteins either as purified preparations or in theirfunction in the subject reconstituted mixture). The term “purified” asused herein preferably means at least 80% by dry weight, more preferablyin the range of 95-99% by weight, and most preferably at least 99.8% byweight, of biological macromolecules of the same type present (butwater, buffers, and other small molecules, especially molecules having amolecular weight of less than 5000, can be present). The term “pure” asused herein preferably has the same numerical limits as “purified”immediately above. “Isolated” and “purified” do not encompass eitherprotein in its native state (e.g. as a part of a cell), or as part of acell lysate, or that have been separated into components (e.g., in anacrylamide gel) but not obtained either as pure (e.g. lackingcontaminating proteins) substances or solutions. The term isolated asused herein also refers to a component protein that is substantiallyfree of cellular material or culture medium when produced by recombinantDNA techniques, or chemical precursors or other chemicals whenchemically synthesized.

[0095] In the subject method, prenylation systems derived from purifiedproteins may have certain advantages over cell lysate based assays.Unlike the reconstituted protein system, the prenylation activity of acell-lysate may not be readily controlled. Measuring kinetic parametersis made tedious by the fact that cell lysates may be inconsistent frombatch to batch, with potentially significant variation betweenpreparations. In vitro evidence indicates that prenyltransferases havethe ability to cross-prenylate CAAX-related sequences, so that famesyltransferase present in a lysate may provide an unwanted kineticparameter. Moreover, cycling of prenylated proteins by guaninenucleotide dissociation inhibitor (GDI)-like proteins in the lysatecould further complicate kinetics of the reaction mixture. Evaluation ofa potential inhibitor using a lysate system is also complicated in thosecircumstances where the lysate is charged with mRNA encoding the GTPasesubstrate polypeptide or GGPTase activity, as such lysates may continueto synthesize proteins active in the assay during the development periodof the assay, and can do so at unpredictable rates. Knowledge of theconcentration of each component of the prenylation system can berequired for each lysate batch, along with the overall kinetic data, inorder to determine the necessary time course and calculate thesensitivity of experiments performed from one lysate preparation to thenext. The use of reconstituted protein mixtures can allow more carefulcontrol of the reaction conditions in the prenylation reaction.

[0096] The purified protein mixture includes a purified preparation ofthe substrate polypeptide and a geranylgeranyl isoprenoid (or analogthereof) under conditions which drive the conjugation of the twomolecules. For instance, the mixture can include a fungal GGPTase Icomplex including RAM2 and CDC43 subunits, a geranylgeranyl diphosphate,a divalent cation, and a substrate polypeptide, such as may be derivedfrom Rho1.

[0097] Furthermore, the reconstituted mixture can also be generated toinclude at least one auxiliary substrate recognition protein, such as aRab escort protein where GGPTase II is the prenylase employed in thereaction mixture.

[0098] Prenylation of the target regulatory protein via an in vitroprenylation system, in the presence and absence of a candidateinhibitor, can be accomplished in any vessel suitable for containing thereactants. Examples include microtitre plates, test tubes, andmicro-centrifuge tubes. In such embodiments, a wide range of detectionmeans can be practiced to score for the presence of the prenylatedprotein.

[0099] In one embodiment of the present assay, the products of aprenylation system are separated by gel electrophoresis, and the levelof prenylated substrate polypeptide assessed, using standardelectrophoresis protocols, by measuring an increase in molecular weightof the target substrate that corresponds to the addition of one or moregeranylgeranyl moieties. For example, one or both of the targetsubstrate and geranylgeranyl group can be labeled with a radioisotopesuch as ³⁵S, ¹⁴C, or ³H, and the isotopically labeled protein bandsquantified by autoradiographic techniques. Standardization of the assaysamples can be accomplished, for instance, by adding known quantities oflabeled proteins which are not themselves subject to prenylation ordegradation under the conditions which the assay is performed.Similarly, other means of detecting electrophoretically separatedproteins can be employed to quantify the level of prenylation of thetarget substrate, including immunoblot analysis using antibodiesspecific for either the target substrate or geranylgeranyl epitopes.

[0100] As described below, the antibody can be replaced with anothermolecule able to bind one of either the target substrate or theisoprenoid. By way of illustration, one embodiment of the present assaycomprises the use of a biotinylated target substrate in the conjugatingsystem. Indeed, biotinylated GGPTase substrates have been described inthe art (c.f. Yokoyama et al. (1995) Biochemistry 34:1344-1354). Thebiotin label is detected in a gel during a subsequent detection step bycontacting the electrophoretic products (or a blot thereof) with astreptavidin-conjugated label, such as a streptavidin linkedfluorochrome or enzyme, which can be readily detected by conventionaltechniques. Moreover, where a reconstituted protein mixture is used(rather than a lysate) as the conjugating system, it may be possible tosimply detect the target substrate and geranylgeranyl conjugates in thegel by standard staining protocols, including coomassie blue and silverstaining.

[0101] In a similar fashion, prenylated and unprenylated substrate canbe separated by other chromatographic techniques, and the relativequantities of each determined. For example, HPLC can be used toquantitate prenylated and unprenylated substrate (Pickett et al. (1995)Analytical Biochem 225:60-63), and the effect of a test compound on thatratio determined.

[0102] In another embodiment, an immunoassay or similar binding assay,is used to detect and quantify the level of prenylated target substrateproduced in the prenylation system. Many different immunoassaytechniques are amenable for such use and can be employed to detect andquantitate the conjugates. For example, the wells of a microtitre plate(or other suitable solid phase) can be coated with an antibody whichspecifically binds one of either the target substrate or geranylgeranylgroups. After incubation of the prenylation system with and without thecandidate agent, the products are contacted with the matrix boundantibody, unbound material removed by washing, and prenylated conjugatesof the target substrate specifically detected. To illustrate, if anantibody which binds the target substrate is used to sequester theprotein on the matrix, then a detectable anti-geranylgeranyl antibodycan be used to score for the presence of prenylated target substrate onthe matrix.

[0103] Still a variety of other formats exist which are amenable to highthrough put analysis on microtitre plates or the like. The prenylationsubstrate can be immobilized throughout the reaction, such as bycross-linking to activated polymer, or sequestered to the well wallsafter the development of the prenylation reaction. In one illustrativeembodiment, a Rho-like GTPase, e.g. a fungal Rho1, Rho2, Cdc42 orRsr1/Bud1, is cross-linked to the polymeric support of the well, theprenylation system set up in that well, and after completion, the wellwashed and the amount of geranylgeranyl sidechains attached to theimmobilized GTPase detected. In another illustrative embodiment, wellsof a microtitre plate are coated with streptavidin and contacted withthe developed prenylation system under conditions wherein a biotinylatedsubstrate binds to and is sequestered in the wells. Unbound material iswashed from the wells, and the level of prenylated target substrate isdetected in each well. There are, as evidenced by this specification, avariety of techniques for detecting the level of prenylation of theimmobilized substrate. For example, by the use of dansylated (describedinfra) or radiolabelled geranylgeranyl diphosphaste in the reactionmixture, addition of appropriate scintillant to the wells will permitdetection of the label directly in the microtitre wells. Alternatively,the substrate can be released and detected, for example, by any of thosemeans described above, e.g. by radiolabel, gel electrophoresis, etc.Reversibly bound substrate, such as the biotin-conjugated substrate setout above, is particularly amenable to the latter approach. In otherembodiments, only the geranylgeranyl moiety is released for detection.For instance, the thioether linkage of the isoprenoid with the substratepeptide sequence can be cleaved by treatment with methyl iodide. Thereleased geranylgeranyl products can be detected, e.g., byradioactivity, HPLC, or other convenient format.

[0104] Other geranylgeranyl derivatives include detectable labels whichdo not interfere greatly with the conjugation of that group to thetarget substrate. For example, in an illustrative embodiment, the assayformat provides fluorescence assay which relies on a change influorescent activity of a group associated with a GGPTase substrate toassess test compounds against a fungal GGPTase. To illustrate, GGPTase-Iactivity can be measured by a modified version of the continuousfluorescence assay described for farnesyl transferases (Cassidy et al.,(1985) Methods Enzymol. 250: 30-43; Pickett et al. (1995) AnalyticalBiochem 225:60-63; and Stirtan et al. (1995) Arch Biochem Biophys321:182-190). In an illustrative embodiment, dansyl-Gly-Cys-Ile-Ile-Leu(d-GCIIL) and the geranylgeranyl diphosphate are added to assay buffer,along with the test agent or control. This mixture is preincubated at30° C. for a few minutes before the reaction is initiated with theaddition of GGPTase enzyme. The sample is vigorously mixed, and analiquot of the reaction mixture immediately transferred to a prewarmedcuvette, and the fluorescence intensity measured for 5 minutes. Usefulexcitation and emission wavelengths are 340 and 486 nm, respectively,with a bandpass of 5.1 nm for both excitation and emissionmonochromators. Generally, fluorescence data are collected with aselected time increment, and the inhibitory activity of the test agentis determined by detecting a decrease in the initial velocity of thereaction relative to samples which lack a test agent.

[0105] In yet another embodiment, the geranylgeranyl transferaseactivity against a particular substrate can be detected in the subjectassay by using a phosphocellulose paper absorption system (Roskoski etal. (1994) Analytical Biochem 222:275-280), or the like. To effectbinding of a peptidyl substrate to phosphocellulose at low pH, severalbasic residues can be added, preferably to the amino-terminal side ofthe CAAX target sequence of the peptide, to produce a peptide with aminimal minimum charge of +2 or +3 at pH less than 2. This follows thestrategy used for the phosphocellulose absorption assay for proteinkinases. In an illustrative embodiment; the transfer of the [H³]geranylgeranyl group from [H³]-geranylgeranyl pyrophosphate to KLKCAILor other acceptor peptides can be measured under conditions similar tothe famesyl transferase reactions described by Reiss et al. (Reiss etal., (1990) Cell 62: 81-88) In an illustrative embodiment, reactionmixtures can be generated to contain 50 mM Tris-HCL (pH 7.5), 50 μMZnCl₂, 20 mM KCl, 1 mM dithiothreitol, 250 μM KLKCAIL, 0.4 μM [H³]geranylgeranyl pyrophosphate, and 10-1000 μg/ml of purified fungalGGPTase protein. After incubation, e.g., for 30 minutes at 37° C.,samples are applied to Whatman P81 phosphocellulose paper strips. Afterthe liquid permeates the paper (a few seconds), the strips are washed inethanol/phosphoric acid (prepared by mixing equal volumes of 95% ethanoland 75 mM phosphoric acid) to remove unbound isoprenoids. The samplesare air dried, and radioactivity can be measured by liquid scintillationspectrometry. Background values are obtained by using reaction mixturewith buffer in place of enzyme.

[0106] An added feature of this strategy is that it produces hydrophilicpeptides that are more readily dissolved in water. Moreover, theprocedure outlined above works equally well for protein substrates (mostproteins bind to phosphocellulose at acidic pH), so should be usefulwhere full length protein, e.g., Rho1 or Cdc42, are utilized as theGGPTase substrate.

[0107] Likewise, a variety of techniques are known in the art foraccessing the activity of a glucan synthase and can be adapted forgenerating drug screening assays designed to detect inhibitors of afungal glucan synthase complex which includes a Rho-like GTPase. Asabove, the cell-free glucan synthesis systems can be utilized in thesubject assay, and include reconstituted protein mixtures and/or celllysates/membrane preparations. Accordingly, in preferred embodiments,the glucan synthesis system is derived from purified proteinpreparations (preferably reconstituted in a lipid formulation) ormembrane preparations derived from a reagent cells, e.g., a cellexpressing a recombinant Rho1/Gsc1 complex. To illustrate, membraneextracts are prepared from selected cells, homogenized with glass beads,and unbroken cells and debris are removed by centrifugation. Thesupernatant fluids are centrifuged at high speed, and the resultingpellets are washed with buffer containing 0.05M potassium phosphate (pH7.5), 0.5 mM DTT, and 1.0 mM PMSF. The washed pellet is resuspended inthe same buffer containing 5% glycerol. This protein extract serves asthe source for β(1-3)-glucan synthase in the enzymatic assays.

[0108] The β(1-3)-glucan synthase reactions can be performed similar tothose described in the art (e.g., Cabib et al. (1987) Methods Enzymol.138:637-642) and the appended examples. Briefly, a reaction mixture isgenerated containing Tris (or other suitable buffer), dithiothretol, KF,glycerol, PMSF, UDP-glucose, guanosine 5′-(γ-S)-triphosphate (GTPγS),UDP-[³H]glucose (Amersham) plus a sample of membrane protein extract.Optionally, α-amylase can be added to reaction mixtures to eliminate thecontribution of [3H]glucose incorporation into glycogen. The reactionsare performed in the presence or absence of the test compound. Followingincubation for a selected time, the [³H]-glucose incorporated intotrichloroacetic acid-insoluble material is collected onto glass fiberfilters and measured using a liquid scintillation counter.

[0109] In still other embodiments of the subject assay, cell-freemixtures can be utilized to identify agents which inhibit the enzymaticactivity of a fungal PKC/GTPase complex such as the PKC1/Rho1 complex.In an exemplary embodiment, the kinase activity of a PKC1/GTPase complexcan assessed by such methods as described in Watanabe et al. (1994) JBiol Chem 269:16829-16836. For instance, phosphorylation reactions canbe initiated by adding reaction cocktail (40 mM MOPS pH 7.5, 10 mMMgCl₂, 1 mM DTT, 50 μM [γ-³²P]ATP [6 μCi/reaction], a substrate peptideand the PKC/GTPase complex, and incubated for the reaction to develop.Reactions can be terminated by adding 4× Laemmli's sample buffer, andthe samples boiled and subjected to SDS/PAGE. After electrophoresis,gels are fixed in 12.5% trichloroacetic acid for 10 min, washed in 10%methanol/10% acetic acid to reduce background, dried, and subjected toautoradiography. Likewise, capillary zone electrophoresis (CZE)techniques can be used to separate and quantitate phosphorylated andunphosphorylated PKC substrate, especially peptide substrates, followingsuch protocols as described by Dawson et al. (1994) Analytical Biochem220: 340-345. Alternatively, reactions can be terminated by spottingonto P81 paper (Whatman). The paper washed three times with 75 mM H₃PO₄and subjected to scintillation counting.

[0110] In another embodiment, the assay is started with the addition ofenzyme and stopped after a set time by the addition of 25%trichloroacetic acid (TCA) and 1.0 mg/ml bovine serum albumin (BSA). Theradioactive product is retained and washed on glass fiber filters thatallow the unreacted ³²P-ATP to pass through. As above, the amount ofphosphorylation is determined by the radioactivity measured in ascintillation counter.

[0111] In still another embodiment, the kinase substrate can beseparated by affinity tags. For instance, a biotinylated peptidesubstrate of the PKC/Rho I complex can be provided in the kinasereaction mixture with [α³²P]ATP, the ³²P label incorporated into thepeptide substrate can be detected by standard scintillation methods. Anadvantage to the biotin-capture system is that it tends to be morequantitative with respect to peptide sequestration relative to, forexample, phosocellulose paper.

[0112] The artificial substrate used can be a synthetic peptideresembling the pseudosubstrate site of PKC1p. All known isoforms of PKCpossess a sequence within their regulatory domains that is related toPKC phosphorylation sites, except for an alanine in place of the targetserine or threonine of a substrate. These sequences, known aspseudosubstrate sites, have been proposed to act as autoinhibitors ofPKC activity. Autoinhibition is thought to be relieved upon binding ofactivating cofactors to the regulatory domain. Peptides resemblingpseudosubstrate sites, except with a serine or threonine in place ofalanine, are known to be excellent substrates for PKC (House et al.(1987) Science 238:1726-1728). Therefore, one substrate that may be usedto test fungal PKC1 complexes is the 15-amino acid peptide,GGLHRHGTIINRKEE, corresponding to residues 394-408 of PKC1p of S.cerevisae (the putative pseudosubstrate site), with a threonine in placeof alanine at position 401.

[0113] Yet another technique which can be used to follow the kinaseactivity of a PKC/GTPase complex in the presence of a test agentinvolves a spectrophotometric assay relying on an ADP produced by thekinase-mediated phosphorylation reaction. Briefly, the formation of ADPin the kinase reaction can be coupled to the pyruvate kinase reaction toproduce pyruvate which is, in turn, coupled to the lactate dehydrogenasereaction with the concomitant oxidation of DPNH to DPN+. The decrease inabsorbance of 340 nm is used to determine the reaction rate. See, forexample, Roskosi (1983) Methods Enzymol-i, 99:3-6.

[0114] In addition to the prenylation and other enzymatic reaction-basedassays, it is contemplated that any of the novel protein-proteininteractions described herein could be directly be the target of a drugscreening assay. For example, in one embodiment, the interaction betweena GTPase and a catalytic subunit of a fungal glucan synthase, such asGsc1/Fsk1 homologs, can be detected in the presence and the absence of atest compound. In another embodiment, the ability of a compound toinhibit the binding of a GTPase protein with a fungal PKC-like protein,such as PKC1, can be assessed in the subject assay. A variety of assayformats for detecting non-enzymatic protein interactions will sufficeand, in light of the present invention, will be comprehended by askilled artisan.

[0115] Complex formation between the GTPase polypeptide and a “targetpolypeptide” (e.g., a PKC polypeptide, a GS subunit, or a GGPTase) maybe detected by a variety of techniques. Modulation of the formation ofcomplexes can be quantitated using, for example, detectably labeledproteins such as radiolabeled, fluorescently labeled, or enzymaticallylabeled GTPase polypeptides, by immunoassay, by chromatographicdetection, or by detecting the intrinsic activity of either the GTPaseor target polypeptide.

[0116] Typically, it will be desirable to immobilize either the GTPaseor the target polypeptide to facilitate separation of complexes fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a GTPase polypeptide tothe target polypeptide, in the presence and absence of a candidateagent, can be accomplished in any vessel suitable for containing thereactants. Examples include microtitre plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase/GTPase (GST/GTPase)fusion proteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtitre plates,which are then combined with a preparation of a target polypeptide, e.g.a labeled target polypeptide, along with the test compound, and themixture incubated under conditions conducive to complex formation, e.g.at physiological conditions for salt and pH, though slightly morestringent conditions may be desired. Following incubation, the beads arewashed to remove any unbound label, and the matrix immobilized andlabeled target polypeptide retained on the matrix determined directly,or in the supernatant after the complexes are subsequently dissociated.Alternatively, the complexes can be dissociated from the matrix,separated by SDS-PAGE, and the level of target polypeptide found in thebead fraction quantitated from the gel using standard electrophoretictechniques.

[0117] Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, either the GTPaseor target polypeptide can be immobilized utilizing conjugation of biotinand streptavidin. For instance, biotinylated GTPase molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, antibodies reactive withGTPase, but which do not interfere with the interaction between theGTPase and target polypeptide, can be derivatized to the wells of theplate, and GTPase trapped in the wells by antibody conjugation. Asabove, preparations of a target polypeptide and a test compound areincubated in the GTPase-presenting wells of the plate, and the amount ofcomplex trapped in the well can be quantitated. Other exemplary methodsfor detecting such complexes, in addition to those described above,include detection of a radiolabel or fluorescent label; immunodetectionof complexes using antibodies reactive with the target polypeptide, orwhich are reactive with GTPase protein and compete with the targetpolypeptide; as well as enzyme-linked assays which rely on detecting anenzymatic activity associated with the target polypeptide, e.g., eitherintrinsic or extrinsic activity. In the instance of the latter, theenzyme can be chemically conjugated or provided as a fusion protein withthe target polypeptide. To illustrate, the target polypeptide can bechemically cross-linked or genetically fused with horseradishperoxidase, and the amount of polypeptide trapped in the complex can beassessed with a chromogenic substrate of the enzyme, e.g.3,3′-diamino-benzadine terahydrochloride or 4-chloro-1-napthol.Likewise, a fusion protein comprising the target polypeptide andglutathione-S-transferase can be provided, and complex formationquantitated by detecting the GST activity using1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).Alternatively, using such substrates as described above, an intrinsicactivity of the target polypeptide can be used to facilitate detection.

[0118] For processes which rely on immunodetection for quantitating oneof the proteins trapped in the complex, antibodies against the targetprotein or GTPase protein, can be used. Alternatively, the protein to bedetected in the complex can be “epitope tagged” in the form of a fusionprotein which includes a second polypeptide for which antibodies arereadily available (e.g. from commercial sources). For instance, the GSTfusion proteins described above can also be used for quantification ofbinding using antibodies against the GST moiety. Other useful epitopetags include myc-epitopes (e.g., see Ellison et al. (1991) J Biol Chem266:21150-21157) which includes a 10-residue sequence from c-myc, aswell as the pFLAG system (International Biotechnologies, Inc.) or thepEZZ-protein A system (Pharamacia, N.J.).

[0119] Cell-based Assay Formats

[0120] In yet further embodiments, the drug screening assay is derivedto include a whole cell expressing a fungal GTPase protein, along withone or more of a GGPTase, a PKC or a glucan synthase catalytic subunit.In preferred embodiments, the reagent cell is a non-pathogenic cellwhich has been engineered to express one or more of these proteins fromrecombinant genes cloned from a pathogenic fungus. For example,non-pathogenic fungal cells, such as S. cerevisae, can be derived toexpress a Rho-like GTPase from a fungal pathogen such as Candidaalbicans. Furthermore, the reagent cell can be manipulated, particularlyif it is a yeast cell, such that the recombinant gene(s) complement aloss-of-function mutation to the homologous gene in the reagent cell. Inan exemplary embodiment, a non-pathogenic yeast cell is engineered toexpress a Rho-like GTPase, e.g. Rho1, and at least one of the subunitsof a GGPTase, e.g. RAM2 and/or Cdc43, derived from a fungal protein. Onesalient feature to such reagent cells is the ability of the practitionerto work with a non-pathogenic strain rather than the pathogen itself.Another advantage derives from the level of knowledge, and availablestrains, when working with such reagent cells as S. cerevisae.

[0121] The ability of a test agent to alter the activity of the GTPaseprotein can be detected by analysis of the cell or products produced bythe cell. For example, agonists and antagonists of the GTPase biologicalactivity can be detected by scoring for alterations in growth orviability of the cell. Other embodiments will permit inference of thelevel of GTPase activity based on, for example, detecting expression ofa reporter, the induction of which is directly or indirectly dependenton the activity of a Rho-like GTPase. General techniques for detectingeach are well known, and will vary with respect to the source of theparticular reagent cell utilized in any given assay.

[0122] For example, quantification of proliferation of cells in thepresence and absence of a candidate agent can be measured with a numberof techniques well known in the art, including simple measurement ofpopulation growth curves. For instance, where the assay involvesproliferation in a liquid medium, turbidimetric techniques (i.e.absorbence/transmittance of light of a given wavelength through thesample) can be utilized. For example, in the instance where the reagentcell is a yeast cell, measurement of absorbence of light at a wavelengthbetween 540 and 600 nm can provide a conveniently fast measure of cellgrowth. Likewise, ability to form colonies in solid medium (e.g. agar)can be used to readily score for proliferation. In other embodiments, aGTPase substrate protein, such as a histone, can be provided as a fusionprotein which permits the substrate to be isolated from cell lysates andthe degree of acetylation detected. Each of these techniques aresuitable for high through-put analysis necessary for rapid screening oflarge numbers of candidate agents.

[0123] Additionally, visual inspection of the morphology of the reagentcell can be used to determine whether the biological activity of thetargeted GTPase protein has been affected by the added agent. Toillustrate, the ability of an agent to create a lytic phenotype which ismediated in some way by a recombinant GTPase protein can be assessed byvisual microscopy.

[0124] The nature of the effect of test agent on reagent cell can beassessed by measuring levels of expression of specific genes, e.g., byreverse transcription-PCR. Another method of scoring for effect onGTPase activity is by detecting cell-type specific marker expressionthrough immunofluorescent staining. Many such markers are known in theart, and antibodies are readily available.

[0125] In yet another embodiment, in order to enhance detection of celllysis, the target cell can be provided with a cytoplasmic reporter whichis readily detectable, either because it has “leaked” outside the cell,or substrate has “leaked” into the cell, by perturbations in the cellwall. Preferred reporters are proteins which can be recombinantlyexpressed by the target cell, do not interfere with cell wall integrity,and which have an enzymatic activity for which chromogenic orfluorogenic substrates are available. In one example, a fungal cell canbe constructed to recombinantly express the β-galactosidase gene from aconstruct (optionally) including an inducible promoter. At some timeprior to contacting the cell with a test agent, expression of thereporter protein is induced. Agents which inhibit prenylation of aRho-like GTPase in the cell, or the subsequent involvement of a Rho-likeGTPase in cell wall integrity, can be detected by an increase in thereporter protein activity in the culture supernatant or from permeationof a substrate in the cell. This, for example, β-galactosidase activitycan be scored using such calorimetric substrates as5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside or fluorescentsubstrates such as methylumbelliferyl-β-D-galactopyranoside. Permeationof the substrate into the cell, or leakage of the reporter into theculture media, is thus readily detectable.

[0126] In yet another embodiment, the alteration of expression of areporter gene construct provided in the reagent cell provides a means ofdetecting the effect on GTPase activity. For example, reporter geneconstructs derived using the transcriptional regulatory sequences, e.g.the promoters, for genes regulated by signal transduction processesdownstream of the target Rho-like GTPase can be used to drive theexpression of a detectable marker, such as a luciferase gene or thelike. In an illustrative embodiment, the construct is derived using thepromoter sequence from a gene expressed in PCK1-dependent heat shockresponse.

[0127] In still another embodiment, the membrane localization resultingfrom prenylation of the fungal GTPase can be exploited to generate thecell-based assay. For instance, the subject assay can be derived with areagent cell having: (i) a reporter gene construct including atranscriptional regulatory element which can induce expression of thereporter upon interaction of the transcriptional regulatory proteinportion of the above fusion protein. For example, a gal4 protein can befused with a Rho1 polypeptide sequence which includes the CAAXprenylation target. Absent inhibitors of GGPTase activity in the reagentcell, prenylation of the fusion protein will result in partitioning ofthe fusion protein at the cell surface membrane. This provides a basallevel of expression of the reporter gene construct. When contacted withan agent that inhibits prenylation of the fusion protein, partitioningis lost and, with the concomitant increase in nuclear concentration ofthe protein, expression from the reporter construct is increased.

[0128] In a preferred embodiment, the cell is engineered such thatinhibition of the GGPTase activity does not result in cell lysis. Forexample, as described in Ohya et al. (1993) Mol Cell Biol 4:1017-1025,mutation of the C-terminus of Rho1 and cdc42 can provide proteins whichare targets of farsenyl transferase rather than geranylgeranyltransferase. As Ohya et al. describe, such mutants can be used to renderthe GGPTase I activity dispensable. Accordingly, providing a reportergene construct and an expression vector for the GGPTasesubstrate/transcription factor fusion protein in such cells as YOT35953cells (Ohya et al., supra) generates a cell whose viability vis-à-visthe GGPTase activity is determined by the reporter construct, if at all,rather than by prenylation of an endogenous Rho-like GTPase by theGGPTase. Of course, the reporter gene product can be derived to have noeffect on cell viability, providing for example another type ofdetectable marker (described, infra). Such cells can be engineered toexpress an exogenous GGPTase activity in place of an endogenousactivity, or can rely on the endogenous activity. To further illustrate,the Call mutant YOT35953 cell can be further manipulated to express aCall homolog from, e.g., a fungal pathogen or a mammalian cell.

[0129] Alternatively, where inhibition of a GGPTase activity causes celllysis and reporter gene expression, the leakage assay provided above canbe utilized to detect expression of the reporter protein. For instance,the reporter gene can encode β-galactosidase, and inhibition of theGGPTases activity scored for by the presence of cells which take upsubstrate due to loss of cell wall integrity, and convert substrate dueto the expression of the reporter gene.

[0130] In preferred embodiments, the reporter gene is a gene whoseexpression causes a phenotypic change which is screenable or selectable.If the change is selectable, the phenotypic change creates a differencein the growth or survival rate between cells which express the reportergene and those which do not. If the change is screenable, the phenotypechange creates a difference in some detectable characteristic of thecells, by which the cells which express the marker may be distinguishedfrom those which do not.

[0131] The marker gene is coupled to GTPase-dependent activity, be itmembrane association, or a downstream signaling pathway induced by aGTPase complex, so that expression of the marker gene is dependent onthe activity of the GTPase. This coupling may be achieved by operablylinking the marker gene to a promoter responsive to the therapeuticallytargeted event. The term “GTPase-responsive promoter” indicates apromoter which is regulated by some product or activity of the fungalGTPase. By this manner, the activity of a GGPTase can be detected by itseffects on prenylation of GTPase and, accordingly, the downstreamtargets of the prenylated protein. Thus, transcriptional regulatorysequences responsive to signals generated by PKC/GTPase, GS/GTPaseand/or other GTPase complexes, or to signals by other proteins in suchcomplexes which are interupted by GTPase binding, can be used to detectfunction of Rho-like GTPases such as Rho1 and cdc42.

[0132] In the case of yeast, suitable positively selectable (beneficial)genes include the following: URA3, LYS2, HIS3, LEU2, TRP1;ADE1,2,3,4,5,7,8; ARG1, 3, 4, 5, 6, 8; HIS1, 4, 5; ILV1, 2, 5; THR1, 4;TRP2, 3, 4, 5; LEU1, 4; MET2,3,4,8,9,14,16,19; URA1,2,4,5,10; HOM3,6;ASP3; CHO1; ARO 2,7; CYS3; OLE1; IN012,4; PR013. Countless other genesare potential selective markers. The above are involved inwell-characterized biosynthetic pathways. The imidazoleglycerolphosphate dehydratase (IGP dehydratase) gene (HIS3) is preferred becauseit is both quite sensitive and can be selected over a broad range ofexpression levels. In the simplest case, the cell is auxotrophic forhistidine (requires histidine for growth) in the absence of activation.Activation of the gene leads to synthesis of the enzyme and the cellbecomes prototrophic for histidine (does not require histidine). Thusthe selection is for growth in the absence of histidine. Since only afew molecules per cell of IGP dehydratase are required for histidineprototrophy, the assay is very sensitive.

[0133] The marker gene may also be a screenable gene. The screenedcharacteristic may be a change in cell morphology, metabolism or otherscreenable features. Suitable markers include beta-galactosidase (Xgal,C₁₂FDG, Salmon-gal, Magenta-Gal (latter two from Biosynth Ag)), alkalinephosphatase, horseradish peroxidase, exo-glucanase (product of yeastexbl gene; nonessential, secreted); luciferase; bacterial greenfluorescent protein; (human placental) secreted alkaline phosphatase(SEAP); and chloramphenicol transferase (CAT). Some of the above can beengineered so that they are secreted (although not β-galactosidase). Apreferred screenable marker gene is beta-galactosidase; yeast cellsexpressing the enzyme convert the colorless substrate Xgal into a bluepigment.

[0134] In another embodiment, the present invention provides acell-based assay which is based on our finding that the Cal1-1 mutant(see Example 3), e.g., a mutant of the GGPTase subunit cdc43, results insupersensitivity to echinocandin. This observation suggests to us thatGGPTase I inhibitors can enhance sensitivity to GS inhibitors, aphenotype which can be easily detected. In an exemplary embodiment, afungal cell can be contacted with a test agent, and a GS inhibitor suchas echinocandin B (other congeners of the echinocandin class of agents,such as cilofungin, certain pneumocandins, and WF11899A, B and C). Theamount of cell lysis is determined and compared to the amount of celllysis is the absence of the GS inhibitor. Synergism, e.g., astatistically significant increase in lysis of the GS inhibitor treatedcell relative to the cell contacted only with the test agent, suggeststhat the test agent is likely to be a cytotoxic agent which targetsprenylation of Rho-like GTPases, or the association of prenylatedRho-like GTPases with proteins critical to cell wall integrity. Thefungal cell can be a wild-type or recombinant cell, e.g., such as an S.cerevisiae cell engineered to express Candida proteins.

[0135] It has also been observed in the art that mutations to Gsc1(Fks1) confer hypersensitivity to the immunosuppressants FK506 andcyclosporin A (Douglas et al. (1994) PNAS 91:12907). The mechanism ofaction of such agents is understood to involve inhibition of expressionof the Fks2 gene (Mazur et al. (1995) Mol Cell Biol 15:5671). Similar tothe echinocandin-sensitivity assay embodiments provided above, anotherassay format provides a cell in which Fks2 activity is compromised.Synergism of the Fks2 impairment with a test compound can be used toidentify inhibitors of, for example, the glucan synthase subunit Gsc1.For instance, FK506 or cyclosporin A can be used to impair Fks2activity, as can mutations to calcineurin or to the Fks2 gene.

[0136] These observations also suggest that Cal1-1 cells or the like,e.g., impaired for certain GGPTase activities, are suitable for use inassay to detect GS inhibitors, as such cells are more sensitive to theeffects of GS inhibitors. The benefits to enhanced sensitivity includespeedier development of assay readouts, and the further prejudicing ofthe assay towards GS inhibitors rather than other targets which may notprovide cytotoxicity. The latter can provide the ability to identifypotential hits which may not themselves be potent GS inhibitors, butwhich can be manipulated, e.g., by combinatorial chemistry approaches,to provide potent and specific GS inhibitors.

[0137] In yet another embodiment, fungal proteins involved in thevarious interactions set out as targets above can be used to generate aninteraction trap assay (see also, U.S. Pat. No. 5,283,317; Zervos et al.(1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; andIwabuchi et al. (1993) Oncogene 8:1693-1696), for subsequently detectingagents which disrupt binding of the proteins to one and other.

[0138] In particular, the method makes use of chimeric genes whichexpress hybrid proteins. To illustrate, a first hybrid gene comprisesthe coding sequence for a DNA-binding domain of a transcriptionalactivator fused in frame to the coding sequence for a “bait” protein,e.g., a fungal Rho1. The second hybrid protein encodes a transcriptionalactivation domain fused in frame to a gene encoding a “fish” proteinwhich interacts with the Rho1 protein, e.g. a Gsc1 protein. If the baitand fish proteins are able to interact, e.g., form a Rho1/Gsc1 complex,they bring into close proximity the two domains of the transcriptionalactivator. This proximity is sufficient to cause transcription of areporter gene which is operably linked to a transcriptional regulatorysite responsive to the transcriptional activator, and expression of thereporter gene can be detected and used to score for the interaction ofthe bait and fish proteins.

[0139] In accordance with the present invention, the method includesproviding a host cell, preferably a yeast cell, most preferablySaccharomyces cerevisiae or Schizosaccharomyces pombe. The host cellcontains a reporter gene having a binding site for the DNA-bindingdomain of a transcriptional activator, such that the reporter geneexpresses a detectable gene product when the gene is transcriptionallyactivated. Such activation occurs when the activation domain of thetranscriptional activator is brought into sufficient proximity to theDNA-binding domain of a transcriptional activator bound to theregulatory element of the reporter gene. The first chimeric gene may bepresent in a chromosome of the host cell, or as part of an expressionvector.

[0140] A first chimeric gene is provided which is capable of beingexpressed in the host cell. The gene encodes a chimeric protein whichcomprises (i) a DNA-binding domain that recognizes the responsiveelement on the reporter gene in the host cell, and (ii) bait protein,such as Rho1.

[0141] A second chimeric gene is provided which is capable of beingexpressed in the host cell. In one embodiment, both the first and thesecond chimeric genes are introduced into the host cell in the form ofplasmids. Preferably, however, the first chimeric gene is present in achromosome of the host cell and the second chimeric gene is introducedinto the host cell as part of a plasmid. The second chimeric geneincludes a DNA sequence that encodes a second hybrid protein comprisinga transcriptional activation domain fused to a fish protein, or afragment thereof, which is to be tested for interaction with the baitprotein. The fish protein can be a subunit of a GGPTase which interactswith Rho1, or a subunit of a glucan synthase which interacts with Rho1,or Pkc1.

[0142] Preferably, the DNA-binding domain of the first hybrid proteinand the transcriptional activation domain of the second hybrid proteinare derived from transcriptional activators having separable DNA-bindingand transcriptional activation domains. For instance, these separateDNA-binding and transcriptional activation domains are known to be foundin the yeast GAL4 protein, and are known to be found in the yeast GCN4and ADR1 proteins. Many other proteins involved in transcription alsohave separable binding and transcriptional activation domains which makethem useful for the present invention, and include, for example, theLexA and VP16 proteins. It will be understood that other (substantially)transcriptionally -inert DNA-binding domains may be used in the subjectconstructs; such as domains of ACE1, λcI, lac repressor, jun or fos. Inanother embodiment, the DNA-binding domain and the transcriptionalactivation domain may be from different proteins. The use of a LexA DNAbinding domain provides certain advantages. For example, in yeast, theLexA moiety contains no activation function and has no known effect ontranscription of yeast genes. In addition, use of LexA allows controlover the sensitivity of the assay to the level of interaction (see, forexample, the Brent et al. PCT publication WO94/10300).

[0143] In preferred embodiments, any enzymatic activity associated withthe bait or fish proteins is inactivated, e.g., dominant negativemutants of Rho1 and the like can be used. Where the interacting proteinsare of the enzyme-substrate relationship, mutation of one or morecatalytic residues of the enzyme can provide a mutant protein whichretains the ability to bind the substrate but not catalytically convertit to product.

[0144] Continuing with the illustrated example, the Rho1/Gsc1-mediatedinteraction, if any, between the first second fusion proteins in thehost cell, therefore, causes the activation domain to activatetranscription of the reporter gene. The method is carried out byintroducing the first chimeric gene and the second chimeric gene intothe host cell, and subjecting that cell to conditions under which thefirst hybrid protein and the second hybrid protein are expressed insufficient quantity for the reporter gene to be activated. The formationof a Rho1/Gsc1 complex results in a detectable signal produced by theexpression of the reporter gene. Accordingly, the formation of a complexin the presence of a test compound to the level of Rho1/GSC1 complex inthe absence of the test compound can be evaluated by detecting the levelof expression of the reporter gene in each case.

[0145] In an illustrative embodiment, Saccharomyces cerevisiae YPB2cells are transformed simultaneously with a plasmid encoding aGAL4db-Rho1 fusion and with a plasmid encoding the GAL4ad domain fusedto a fungal Gsc1 gene. Moreover, the strain is transformed such that theGAL4-responsive promoter drives expression of a phenotypic marker. Forexample, the ability to grow in the absence of histidine can depend onthe expression of the LacZ gene. When the LacZ gene is placed under thecontrol of a GAL4-responsive promoter, the yeast cell will turn blue inthe presence of β-gal if a functional GAL4 activator has beenreconstituted through the interaction of Rho1 and Gsc1. Thus, aconvenient readout method is provided. Other reporter constructs will beapparent, and include, for example, reporter genes which produce suchdetectable signals as selected from the group consisting of an enzymaticsignal, a fluorescent signal, a phosphorescent signal and drugresistance.

[0146] A similar method modifies the interaction trap system byproviding a “relay gene” which is regulated by the transcriptionalcomplex formed by the interacting bait and fish proteins. The geneproduct of the relay gene, in turn, regulates expression of a reportergene, the expression of the latter being what is scored in the modifiedITS assay. Fundamentally, the relay gene can be seen as a signalinverter.

[0147] As set out above, in the standard ITS, interaction of the fishand bait fusion proteins results in expression of a reporter gene.However, where inhibitors of the interaction are sought, a positivereadout from the reporter gene nevertheless requires detectinginhibition (or lack of expression) of the reporter gene.

[0148] In the inverted ITS system, the fish and bait proteins positivelyregulate expression of the relay gene. The relay gene product is in turna repressor of expression of the reporter gene. Inhibition of expressionof the relay gene product by inhibiting the interaction of the fish andbait proteins results in concomitant relief of the inhibition of thereporter gene, e.g., the reporter gene is expressed. For example, therelay gene can be the repressor gene under control of a promotersensitive to the Rho1/Gsc1 complex described above. The reporter genecan accordingly be a positive signal, such as providing for growth(e.g., drug selection or auxotrophic relief), and is under the controlof a promoter which is constitutively active, but can be suppressed bythe repressor protein. In the absence of an agent which inhibits theinteraction of the fish and bait protein, the repressor protein isexpressed. In turn, that protein represses expression of the reportergene. However, an agent which disrupts binding of the Rho1 and Gsc1proteins results in a decrease in repressor expression, and consequentlyan increase in expression of the reporter gene as repression isrelieved. Hence, the signal is inverted.

[0149] Returning to the teachings of Ohya et al. (1993) Mol Cell Biol4:1017-1025, it is noted that there are only two essential targets ofGGPTase in S. cerevisae, the Rho-like GTPases Rho1 and cdc42. With suchobservations in mind, yet another embodiment of the subject assayutilizes a side-by-side comparison of the effect of a test agent on (i)a cell which prenylates a Rho-like GTPase by adding geranylgeranylmoieties, and (ii) a cell which prenylates an equivalent Rho-like GTPaseby adding farnesyl moieties. In particular, the assay makes use of theability to suppress GGPTase I defects in yeast by altering theC-terminal tail of Rho1 and cdc42 to become substrate targets offarnesyl transferase (see Ohya et al., supra). According to the presentembodiment, the assay is arranged by providing a yeast cell in which thetarget Rho-like GTPases is prenylated by a GGPTase activity of the cell.Both the GGPTase and GTPase can be endogenous to the “test” cell, or oneor both can be recombinantly expressed in the cell. The level ofprenylation of the GTPase is detected, e.g., cell lysis or other meansdescribed above. The ability of the test compound to inhibit theaddition of geranylgeranyl groups to the GTPase in the first cell iscompared against the ability of test compound to inhibit thefarnesylation of the GTPase in a control cell. The “control” cell ispreferably identical to the test cell, with the exception that thetargeted GTPase(s) are mutated at their CAAX sequence to becomesubstrates for FPTases rather than GGPTases. Agents which inhibitprenylation in the test cell but not the control cell are selected aspotential antifungal agents. Such differential screens can beexquisitely sensitive to inhibitors of GGPTase I prenylation of Rho-likeGTPases. In a preferred embodiment, the test cell is derived from the S.cerivisae cell YOT35953 (Ohya et al., supra) or the like which isdefective in GGPTase subunit cdc43. The cell is then engineered with acdc43 subunit from a fungal pathogen such as Candida albicans togenerate the test cell, and additionally with the mutated Rho-likeGTPases to generate the control cell.

[0150] Differential Screening Formats

[0151] In a preferred embodiment, assays can be used to identifycompounds that have therapeutic indexes more favorable than suchantifungal as, for example, papulacandins or echinocandins or the like.For instance, antifungal agents can be identified by the present assayswhich inhibit proliferation of yeast cells or other lower eukaryotes,but which have a substantially reduced effect on mammalian cells,thereby improving therapeutic index of the drug as an anti-mycoticagent.

[0152] In one embodiment, the identification of such compounds is madepossible by the use of differential screening assays which detect andcompare the ability of the test compound to inhibit an activityassociated with a fungal GTPase, relative to its ability to inhibit ananalogous activity of a human GTPase. To illustrate, the assay can bedesigned for side-by-side comparison of the effect of a test compound onthe prenylation activity or protein interactions of fungal and humanGGPTase and GTPase proteins. Given the apparent diversity of GGPTaseproteins, it is probable that the fungal and human GGPTases differ bothin substrate specificity and mechanistic action which can be exploitedin the subject assay. Running the fungal and human prenylation systemsside-by-side permits the detection of agents which have a greaterinhibitory effect (e.g. statistically significant) on the prenylationreaction mediated by the fungal GGPTase than the human enzyme.

[0153] Accordingly, differential screening assays can be used to exploitthe difference in protein interactions and/or catalytic mechanism ofmammalian and fungal GGPTases in order to identify agents which displaya statistically significant increase in specificity for inhibiting thefungal prenylation reaction relative to the mammalian prenylationreaction. Thus, lead compounds which act specifically on the prenylationreaction in pathogens, such as fungus involved in mycotic infections,can be developed. By way of illustration, the present assays can be usedto screen for agents which may ultimately be useful for inhibiting thegrowth of at least one fungus implicated in such mycosis as candidiasis,aspergillosis, mucormycosis, blastomycosis, geotrichosis,cryptococcosis, chromoblastomycosis, coccidioidomycosis,conidiosporosis, histoplasmosis, maduromycosis, rhinosporidosis,nocaidiosis, para-actinomycosis, penicilliosis, monoliasis, orsporotrichosis. For example, if the mycotic infection to which treatmentis desired is candidiasis, the present assay can comprise comparing therelative effectiveness of a test compound on inhibiting the prenylationof a mammalian GTPase protein with its effectiveness towards inhibitingthe prenylation of a GTPase from a yeast selected from the groupconsisting of Candida albicans, Candida stellatoidea, Candidatropicalis, Candida parapsilosis, Candida krusei, Candidapseudotropicalis, Candida quillermondii, or Candida rugosa. Likewise,the present assay can be used to identify anti-fungal agents which mayhave therapeutic value in the treatment of aspergillosis by selectivelytargeting, relative to human cells, GTPase homologs from yeast such asAspergillus fumigatus, Aspergillus flavus, Aspergillus niger,Aspergillus nidulans, or Aspergillus terreus. Where the mycoticinfection is mucormycosis, the GTPase system to be screened can bederived from yeast such as Rhizopus arrhizus, Rhizopus oryzae, Absidiacorymbifera, Absidia ramosa, or Mucor pusillus. Sources of other assayreagents for includes the pathogen Pneumocystis carinii.

[0154] Thus, it is also deemed to be within the scope of this inventionthat the recombinant GTPase cells of the present assay can be generatedso as to comprise heterologous GTPase proteins from metazoan sourcessuch as humans (i.e. cross-species expression). For example, GTPaseproteins from humans can be expressed in the reagent cells underconditions wherein the heterologous protein is able to rescueloss-of-function mutations in the host cell. For example, the reagentcell can be a yeast cell in which a human GTPase protein (e.g.exogenously expressed) is to be a counter-screen for identifying agentswhich selectively inhibit yeast GTPase activities. To illustrate, theYOC706 strain, described by Qadota et al. (1994) Genetics 91:9317-9321,lacks a functional endogenous Rho1 gene, and can be transfected with anexpression plasmid including a human GTPase gene such as RHoA in orderto complement the Rho1 loss-of-function. For example, the codingsequence for RHoA can be cloned into a pRS integrative plasmidcontaining a selectable marker (Sikorski et al. (1989) Genetics122:19-27), and resulting construct used to transform the YOC706 strain.The resulting cells should produce a human RHoA protein which is capableof performing at least some of the functions of the yeast Rho1 protein.The GTPase transformed yeast cells can be easier to manipulate thanmammalian cells, and can also provide access to certain assay formats,such as turbidity detection, which may not be obtainable with mammaliancells.

[0155] Reagents

[0156] If yeast cells are used, the yeast may be of any species whichare cultivable and, preferably, in which an exogenous Rho1-like proteincan be made to engage the appropriate prenylation enzyme and/orparticipate in protein complexes such as with glucan synthesase subunitsor PKC homologs of the host cell. Suitable species include Kluyvereilactis, Schizosaccharomyces pombe, and Ustilaqo maydis; Saccharomycescerevisiae is preferred. Other yeast which can be used in practicing thepresent invention. The term “yeast”, as used herein, includes not onlyyeast in a strictly taxonomic sense, i.e., unicellular organisms, butalso yeast-like multicellular fungi or filamentous fungi.

[0157] The choice of appropriate host cell can be influenced by thechoice of detection signal. For instance, reporter constructs, asdescribed below, can provide a selectable or screenable trait upontranscriptional activation (or inactivation) in response to a signalprovided by the GTPase target. Suitable genes and promoters can bedependent on the reagent cell. Likewise, ease of complementation,genetic manipulation, etc., may also affect the choice of reagent cell.

[0158] With respect to sources for constituting recombinant proteins ofthe subject assays, various GGPTases, GTPases, glucan synthase subunits,and PKC homologs have been identified from a variety of fungal species,and in a significant number of instances, have been cloned so thatrecombinant sources exist.

[0159] For example, identification of enzymes involved in theprenylation pathway from different sources have facilitated the cloningof corresponding genes. For instance, genes GGPTase enzymes, PKChomologs and GTPase homologs have been cloned from various fungalorganisms, and are generally described in the literature and availableon GenBank or other such databases. Complementation of defects in yeastcells such as S. cereviae also constitute a standard protocol forisolating genes encoding fungal and mammalian homologs (as appropriate)of such target proteins as GGPTase subunits, Rho-like GTPases, PKChomologs and glucan synthase subunits.

[0160] The proteins provided in the subject assay can be derived bypurification from a cell in which it is endogenously expressed, or froma recombinant source of the protein. In each instance where arecombinant source of a protein is used in the subject assay, themanipulation of the gene encoding the protein and the subsequentexpression of the protein can be carried out by standard molecularbiological techniques. Ligating the polynucleotide sequence encoding therecombinant protein into a gene construct, such as an expression vector,and transforming or transfecting into host cells, either eukaryotic(yeast, avian, insect or mammalian) or prokaryotic (bacterial cells),are standard procedures used in producing other well-known proteins,including the S. cerevisae proteins PCK1, GGPTase, Rho1 and the like.Similar procedures, or obvious modifications thereof, can be employed toprepare and purify recombinant proteins of the prenylation system fromother sources.

[0161] The recombinant protein can be produced by ligating the clonedgene, or a portion thereof, into a vector suitable for expression ineither prokaryotic cells, eukaryotic cells, or both. Expression vehiclesfor production of recombinant proteins include plasmids and othervectors. For instance, suitable vectors for the expression of theseproteins include plasmids of the types: pBR322-derived plasmids,pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids andpUC-derived plasmids for expression in prokaryotic cells, such as E.coli.

[0162] In general, it will be desirable that the gene construct becapable of replication in the host cell. It may be a DNA which isintegrated into the host genome, and thereafter is replicated as a partof the chromosomal DNA, or it may be DNA which replicates autonomously,as in the case of a plasmid. In the latter case, the vector will includean origin of replication which is functional in the host. In the case ofan integrating vector, the vector may include sequences which facilitateintegration, e.g., sequences homologous to host sequences, or encodingintegrases.

[0163] Appropriate cloning and expression vectors for use withbacterial, fungal, yeast, and mammalian cellular hosts are known in theart, and are described in, for example, Powels et al. (Cloning Vectors:A Laboratory Manual, Elsevier, N.Y., 1985). Mammalian expression vectorsmay comprise non-transcribed elements such as an origin of replication,a suitable promoter and enhancer linked to the gene to be expressed, andother 5′ or 3′ flanking nontranscribed sequences, and 5′ or 3′nontranslated sequences, such as necessary ribosome binding sites, apoly-adenylation site, splice donor and acceptor sites, andtranscriptional termination sequences.

[0164] The preferred mammalian expression vectors contain bothprokaryotic sequences, to facilitate the propagation of the vector inbacteria, and one or more eukaryotic transcription units that areexpressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV,pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo andpHyg derived vectors are examples of mammalian expression vectorssuitable for transfection of eukaryotic cells. Some of these vectors aremodified with sequences from bacterial plasmids, such as pBR322, tofacilitate replication and drug resistance selection in both prokaryoticand eukaryotic cells. Alternatively, derivatives of viruses such as thebovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo,pREP-derived and p205) can be used for transient expression of proteinsin eukaryotic cells. The various methods employed in the preparation ofthe plasmids and transformation of host organisms are well known in theart. For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, seeMolecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritschand Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and17.

[0165] A number of vectors exist for the expression of recombinantproteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, andYRP17, as well as the pRS vectors, e.g., pRS303, pRS304, pRS305, pRS306,etc., are cloning and expression vehicles useful in the introduction ofgenetic constructs into S. cerevisiae (see, for example, Broach et al.(1983) in Experimental Manipulation of Gene Expression, ed. M. InouyeAcademic Press, p. 83, incorporated by reference herein). These vectorscan replicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused.

[0166] Moreover, when yeast are used as the reagent cell, it will beunderstood that the expression of a gene in a yeast cell requires apromoter which is functional in yeast. Suitable promoters include thepromoters for gall, metallothionein, 3-phosphoglycerate kinase (Hitzemanet al, J. Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes (Hesset al, J. Adv. Enzyme Req. 7, 149 (1968); and Holland et al.Biochemistry 17, 4900 (1978)), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phospho-fructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phospho-glucose isomerase, and glucokinase. Suitable vectors andpromoters for use in yeast expression are further described in R.Hitzeman et al, EPO Publn. No. 73,657. Other promoters, which have theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, and the aforementioned metallothionein and glyceraldehyde-3-phosphate dehydrogenase, as well as enzymes responsible for maltoseand galactose utilization. Finally, promoters that are active in onlyone of the two haploid mating types may be appropriate in certaincircumstances. Among these haploid-specific promoters, the pheromonepromoters MFa1 and MFα1 are of particular interest.

[0167] In some instances, it may be desirable to derive the host cellusing insect cells. In such embodiments, recombinant polypeptides can beexpressed by the use of a baculovirus expression system. Examples ofsuch baculovirus expression systems include pVL-derived vectors (such aspVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1),and pBlueBac-derived vectors (such as the β-gal containing pBlueBacIII).

[0168] Furthermore, the recombinant protein can be encoded by a fusiongene created to have additional sequences coding for a polypeptideportion of a fusion protein which would facilitate its purification. Forinstance, a fusion gene coding for a purification leader sequencecomprising a poly-(His)/enterokinase cleavage site sequence can beengineered at the a terminus of the protein, thereby enablingpurification of the expressed fusion protein by affinity chromatographyusing a Ni²⁺ metal resin. The purification leader sequence can then besubsequently removed by treatment with enterokinase (e.g., see Hochuliet al. 1987 J. Chromatography 411:177; and Janknecht et al. PNAS88:8972).

[0169] Exemplary Construction of the Expression Plasmid For RecombinantGGPTase-I.

[0170] Polymerase chain reaction (PCR) can be carried out to isolate theCDC43 coding sequence from S cerevisiae. Using a sense strand primer(5′-CCATCGATCATATGTGTCAAGCTAGGAAT-3′) can introduce a unique ClaIrestriction site upstream of the CDC43 start codon and an NdeI site thatoverlaps the ATG initiation codon. An antisense strand PCR primer(5′-GCGGGTACCCTGCAGTCAAAAACAGCACCTTTT-3′) introduces unique PstI andKpnI restriction sites downstream of the CDC43 stop codon. The PCRproduct is ligated into a convenient vector, such as bluescript IISK-(+) using ClaI and KpnI. An XbaI-ClaI fragment containing RAM2 (Mayeret al., (1993) Gene 132:41-47) can be cloned into the CDC43 containingvector, upstream of the CDC43 sequence, to produce a bicistronicconstruct. The RAM2 and CDC43 orfs are then coupled by deletionmutagenesis with the antisense strand primer(5′-GGTAGCTTGAVACATCAAAACTCCTCCTGCAGATTTATTTTG-3′), which overlaps theRAM2 translation termination codon with the CDC43 initiation codon. TheRAM2-CDC43 cassette can then be cloned into an appropriate expressionvector and used to transform E coli.

[0171] Recombinant GGPTase-I can be purified from the resulting culturesas described for recombinant yeast FPTase (Mayer et al., supra), withminor modifications (Stirtan et al. (1995) Arch Biochem Biophys321:182-190). Wet cell paste is resuspended in 16 ml of lysis buffer (50mM Tris-HCl, pH 7.0, 10 mM BME, 10 mM MgCl₂, 50 μM ZnCl₂, 1 mM PMSF) anddisrupted by sonication. The cell-free homogenate is clarified bycentrifugation and chromatographed on DE52 ion-exchange resin (1.5×14cm) at 4° C., preequilibrated with low-salt buffer (50 mM Tris-Hcl, pH7.0, 10 mM MgCl₂, 50 μM ZnCl₂, 10 mM BME). Protein is eluted with astepwise gradient of 0 to 800 mM NaCl in low-salt buffer. RecombinantPGGPTase-I is expected to elute at 200 mM NaCl. The DE52-purifiedmaterial is dialyzed at 4° C. against low-salt buffer, diluted to ˜1mg/ml with the same buffer, and loaded onto an anti-α-tubulinimmunoaffinity column (Mayer et al., supra) preequilibrated with bindingbuffer (20 mM Tris-HCl, pH 7.5, 1 mM MgCl₂, 10 μM ZnCl₂, 5 mM BME, 50 mMNaCl). The column is washed with binding buffer (˜25 ml) and then elutedwith binding buffer containing 5 mM Asp-Phe. Fractions containingGGPTase-I activity are combined. Recombinant GGPTase-I has beendemonstrated to be stable for several months at −80° C. and for severaldays at 0° C.

[0172] Preparation of Dansyl-Gly-Cys-Ile-Ile-Leu.

[0173] Dansyl-Gly-Cys-Ile-Ile-Leu is prepared essentially as describedpreviously for dansyl-Gly-Cys-Val-Ile-Ala (Cassidy et al., (1985)Methods Enzymol. 250: 30-43), the farnesylated substrate correspondingto Cys-Val-Ile-Ala. Dansyl-Gly-Cys-Ile-Ile-Leu can be purified bypreparative HPLC on a Vydac protein and peptide C18 reversed-phasecolumn (22 mm×25 cm) by elution with a gradient of 85-92% CH₃CN/0.1% TFAin H₂O/0.1% TFA over 20 min, followed by a gradient of 92-100%CH₃CN/0.1% TFA over 5 min, and finally with 100% CH₃CN/0.1% TFA for 10min. Organic materials are removed by rotary evaporation, and theresulting aqueous suspension is lyophilized to afforddansyl-Gly-Cys-Ile-Ile-Leu.

[0174] Pharmaceutical Preparations of Identified Agents

[0175] After identifying certain test compounds as potential antifungalagents, the practioner of the subject assay will continue to test theefficacy and specificity of the selected compounds both in vitro and invivo. Whether for subsequent in vivo testing, or for administration toan animal as an approved drug, agents identified in the subject assaycan be formulated in pharmaceutical preparations for in vivoadministration to an animal, preferably a human.

[0176] The subject compounds selected in the subject, or apharmaceutically acceptable salt thereof, may accordingly be formulatedfor administration with a biologically acceptable medium, such as water,buffered saline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like) or suitable mixtures thereof. Theoptimum concentration of the active ingredient(s) in the chosen mediumcan be determined empirically, according to procedures well known tomedicinal chemists. As used herein, “biologically acceptable medium”includes any and all solvents, dispersion media, and the like which maybe appropriate for the desired route of administration of thepharmaceutical preparation. The use of such media for pharmaceuticallyactive substances is known in the art. Except insofar as anyconventional media or agent is incompatible with the activity of thecompound, its use in the pharmaceutical preparation of the invention iscontemplated. Suitable vehicles and their formulation inclusive of otherproteins are described, for example, in the book Remington'sPharmaceutical Sciences (Remington's Pharmaceutical Sciences. MackPublishing Company, Easton, Pa., USA 1985). These vehicles includeinjectable “deposit formulations”. Based on the above, suchpharmaceutical formulations include, although not exclusively, solutionsor freeze-dried powders of the compound in association with one or morepharmaceutically acceptable vehicles or diluents, and contained inbuffered media at a suitable pH and isosmotic with physiological fluids.In preferred embodiment, the compound can be disposed in a sterilepreparation for topical and/or systemic administration. In the case offreeze-dried preparations, supporting excipients such as, but notexclusively, mannitol or glycine may be used and appropriate bufferedsolutions of the desired volume will be provided so as to obtainadequate isotonic buffered solutions of the desired pH. Similarsolutions may also be used for the pharmaceutical compositions ofcompounds in isotonic solutions of the desired volume and include, butnot exclusively, the use of buffered saline solutions with phosphate orcitrate at suitable concentrations so as to obtain at all times isotonicpharmaceutical preparations of the desired pH, (for example, neutralpH).

Exemplification

[0177] The invention, now being generally described, will be morereadily understood by reference to the following examples, which areincluded merely for purposes of illustration of certain aspects andembodiments of the present invention and are not intended to limit theinvention.

EXAMPLE 1 Activation of Yeast Protein Kinase C by Rho1 GTPase

[0178] The abbreviations used in Example 1 are: PKC, protein kinase C;MAPK, mitogen-activated protein kinase; MEK, MAPK-activating kinase;MEKK, MEK-activating kinase; DAG, diacylglycerol; SRF, serum responsefactor; JNK, Jun NH₂-terminal kinase (also known as SAPK,stress-activated protein kinase); PCR, polymerase chain reaction; HA,influenza hemagglutinin; PAGE, polyacrylamide gel electrophoresis; GST,glutathione-S-transferase; PS, phosphatidylserine; PMA, phorbolmyristate acetate; GS, 1,3-β-glucan synthase; MBP, myelin basic protein.

[0179] A. Overview

[0180] We have investigated the role of the essential Rho1 GTPase incell integrity signaling in budding yeast. Conditional rho1 mutantsdisplay a cell lysis defect that is similar to that of mutants in thecell integrity signaling pathway mediated by protein kinase C (PKC1),which is suppressed by overexpression of PKC1. rho1 mutants are alsoimpaired in pathway activation in response to growth at elevatedtemperature. PKC1 co-immuneprecipitates with Rho1 in yeast extracts, andrecombinant Rho1 associates with PKC1 in vitro in a GTP-dependentmanner. Recombinant Rho1 confers upon PKC1 the ability to be stimulatedby phosphatidylserine (PS), indicating that Rho1 controls signaltransmission through PCK1.

[0181] The PKC1 gene of the budding yeast Saccharomyces cerevisiaeencodes a homolog of mammalian protein kinase C (PKC) (ref. 1) thatregulates a MAP kinase (MAPK)-activation cascade comprised of a MEKK(Bck1), a redundant pair of MEKs (Mkk1/2), and a MAPK (Mpk1) (2, 3).Mutants in this signaling cascade, called the cell integrity pathway,undergo cell lysis resulting from a deficiency in cell wall constructionthat is exacerbated by growth at elevated temperatures. We have reportedthat thermal stress activates the cell integrity pathway, and proposedthat weakness in the cell wall that develops during growth at hightemperature induces the signal for pathway activation (4).

[0182] PCK1 most closely resembles the conventional isoforms ofmammalian PKC, which require phospholipids, Ca²⁺, and diacylglycerol(DAG) as cofactors to stimulate their catalytic activity (1). However,in vitro studies of this yeast protein kinase have failed to demonstratestimulation by cofactors, despite the finding that mutations in PKC1predicted to relieve cofactor dependence have an activating effect onthe enzyme (5, 6). This suggested that one or more components requiredfor cofactor-dependent stimulation of PCK1 was missing from in vitroreconstitution experiments.

[0183] Members of the Rho family of small GTPases (RhoA, Cdc42, and Rac)regulate various aspects of actin cytoskeleton organization andactivation of the SRF transcription factor in mammalian cells (7-10).Cdc42 and Rac, but not RhoA, stimulate the signaling pathway thatcontains the JNK/SAPK (Jun NH₂-terminal kinase or stress-activatedprotein kinase) MAPK homolog in mammalian cells (11-13). Downstreameffectors of RhoA have not been identified (14, 15). The yeast RHO1 geneencodes a homolog of mammalian RhoA that resides at sites of cell growth(16) and whose function is essential for viability (17). A rho1Δ mutantis partially suppressed by expression of human RhoA, but a residual celllysis defect is apparent at high temperature (18), suggesting that RHO1may function within the cell integrity pathway. Additionally, anactivated allele of PKC1 was isolated recently as a dominant mutationalsuppressor of this defect (19), further supporting the notion that thesesignaling molecules act through a common pathway. In this communication,we demonstrate that Rho1 associates with PKC1 in a GTP-dependent manner,and confers upon this protein kinase the ability to respond tophosphatidylserine as an activating cofactor.

[0184] B. Experimental Procedures

[0185] Yeast strains and mutant construction—All strains used in thisstudy were derived from YPH500 (See reference of Example 3). Error-pronePCR (21) was used to introduce random mutations into the RHO1 sequence.The PCR-amplified RHO1 fragment was inserted into the EcoRI/BglII gap ofpYO701, and introduced into yeast strain YOC706, which harbors a rho1Δand a plasmid expressing RHO1 under the control of the GAL1 promoter(18). We examined 4000 transformants for growth on YPD (yeastextract/peptone/dextrose) plates at 23° C. and 37° C., and identified 41rho1^(ts) mutations. Among these, 11 rho1 alleles (designatedrho1-1-rho1-11) contained single or double base changes. All of thesealleles were reconstructed by site-directed mutagenesis, and integratedat the ADE3 locus (See reference of Example 3) of diploid strain YOC701(RHO1/rho1Δ::HIS3). Haploid strains used in this study (YOC764 [RHO1],YOC729 [rho1-3], and YOC755 [rho1-5]) were derived from YOC701integrants by standard genetic techniques. A single copy plasmid(pYO904) that carries HA-tagged RHO1 was constructed in vector pRS314,as described previously (16), and introduced into yeast strain YOC701. Asegregant bearing rho1Δ::HIS3 and pYO904, and a wild-type (RHO1)segregant lacking the plasmid were used for coimmunoprecipitationexperiments.

[0186] Antibodies, extracts, immunoprecipitation, protein kinase assaysand immunodetection—Anti-HA antibodies (12CA5; BAbCo, Inc.) were usedfor immunoprecipitation and immunodetection of ^(HA)Rho1, Mpk1^(HA), andPKC1^(HA). Polyvalent PKC1 antibodies (used for immunodetection of PKC1)were raised by Cocalico Biologicals (Reamstown, Pa.) in New Zealandwhite rabbits against a TrpE::PKC1 fusion protein that contains aminoacids 470-664 of PKC1. This antiserum was used (at 1:3000 dilution) forimmunodetection of PKC1. Secondary antibodies used were horseradishperoxidase-conjugated donkey anti-rabbit (Amersham; at 1:10,000dilution).

[0187] Yeast extract preparation, immunoprecipitation, immunodetectionand protein kinase assays of Mpk1^(HA) were conducted as describedpreviously (4). Preparation of cell extracts and immunoprecipitationsfor experiments with ^(HA)Rho1 were carried out as in (4) with somemodifications. Lysis buffer without p-nitrophenyl phosphate and with 1%NP-40 was used. The extract (700 μg protein) was precleared byincubation with 20 μl of a 50% suspension of protein A-sepharose for 1 hprior to immunoprecipitation to eliminate non-specific binding ofproteins to immunecomplexes. Beads were boiled in SDS-PAGE samplebuffer, and samples were applied to 7.5% (for PKC1 blots) or 15% (for^(HA)Rho1 blots) SDS-PAGE gels. For PKC1 kinase assays, all as describedpreviously (5), except for the addition of recombinant GTPases (seebelow). A synthetic peptide corresponding to the sequence surroundingSer939 of Bck1, a phosphorylation site for PKC 1, was used as substratein PKC1 kinase assays (5).

[0188] Recombinant Rho1 and Cdc42. Recombinant GST-Rho1 and GST-Cdc42were expressed and purified from baculovirus-infected insect (Sf9)cells, as described (23). For in vitro association with PKC1^(HA),GST-Rho1 was not eluted from the glutathione agarose beads used forpurification. GST-Rho1-bound beads were incubated with cell extract inimmunoprecipitation buffer (4) for 5 h at 4° C., followed by 3 washeswith this buffer. For use in PKC1^(HA) protein kinase assays, GST-Rho1and GST-Cdc42 were eluted from the beads with reduced glutathione.Purified GST-Rho1 displayed no protein kinase activity against the Bck1peptide in the absence of PKC1 (not shown).

[0189] C. Results and Discussion

[0190] To examine the role of RHO1 in the cell integrity signalingpathway, we isolated a set of 11 temperature-sensitive rho1 alleles byin vitro random mutagenesis. Some of these mutants displayed cell lysisdefects at the restrictive temperature (eg. rho1-5), but others did not(eg. rho1-3; FIG. 1A). Additionally, overexpression of PKC1 suppressedexclusively rho1-5 (FIG. 1B). Because of this allele-specific behavior,we chose rho1-3 and rho1-5 for further study.

[0191] The Mpk1 MAPK is activated via PKC1 in response to brief heatshock treatment (4). To determine if RHO1 is required for cell integritypathway signaling, we tested the ability of rho1^(ts) mutants toactivate Mpk1 upon heat shock. Mpk1, tagged at its COOH-terminus withthe influenza hemagglutinin (HA) epitope (Mpk1 HA), wasimmunoprecipitated from extracts of heat shock-treated cells, andassayed for protein kinase activity in vitro using myelin basic protein(MBP) as substrate. Heat shock-induced activation of Mpk1 was completelyblocked in the rho1-3 mutant (FIG. 2), indicating that RHO1 function isessential for Mpk1 activation. The rho1-5 mutant allowed some Mpk1activation, suggesting that this allele retains some function atrestrictive temperature. Residual function of the rho1-5 allele at hightemperature might also explain the allele-specific suppression of thismutant by PKC1 overexpression if Rho1 function is required for PCK1activation.

[0192] The yeast Cdc42 GTPase interacts with and stimulates the Ste20protein kinase, which regulates the MAPK-activation cascade of the yeastpheromone response pathway (24, 25). Additionally, both recombinanthuman Cdc42 and Rac stimulate a mammalian protein kinase that is closelyrelated to Ste20 (PAK65) (26, 27). Because Ste20 and PKC1 function atanalogous positions in their respective MAPK signaling pathways (2, 3),we examined the possibility that Rho1 interacts directly with PKC1 invivo. Rho1, tagged at its NH₂-terminus with the HA epitope (^(HA)Rho1),was immunoprecipitated from yeast extracts, and the resultantimmunoprecipitates were analyzed by SDS-PAGE and immunoblotting withanti-PKC1 antibody. PKC1 was co-immunoprecipitated with ^(HA)Rho1 (FIG.3A, lanes 4 and 6), suggesting that PKC1 associates with Rho1 in vivo.This interaction was observed both in cells growing at 23° C. and afterheat shock.

[0193] To determine if the association between Rho1 and PKC1 depends onthe activation state of Rho1, we examined the effect of differentguanine nucleotides on this interaction in vitro. Recombinantglutathione-S-transferase-(GST)-Rho1, immobilized on glutathione agarosebeads, was preloaded with either GTPγS or GDP prior to incubation with ayeast extract containing soluble PKC1 tagged at its COOH-terminus withthe HA epitope (PKC1^(HA)). After washing the beads, bound PKC1^(HA) wasdetected by SDS-PAGE and immunoblotting with anti-HA antibody. FIG. 3Bshows that GTPγS-bound GST-Rho1 associated with PCK1 (lane 5), butGDP-bound protein did not (lane 3).

[0194] We also tested the possibility that PKC1 activity is stimulatedby Rho1. PKC1^(HA) was immunoprecipitated from yeast extracts, and itsprotein kinase activity was measured in the presence or absence ofGST-Rho1 using a synthetic Bck1 peptide as substrate. FIG. 4A shows thatGST-Rho1 did not stimulate PKC1 activity alone but, when bound to GTPγS,conferred upon the protein kinase the ability to respond to activatingcofactors (PS, DAG, and Ca²⁺). This stimulatory effect is specific toRho1, because GST-Cdc42 did not confer cofactor-dependent stimulation onPCK1. In the presence of GTP-bound GST-Rho1, PCK1 was strongly activatedby phosphatidylserine (PS) as a lone cofactor (FIG. 4B). Theconventional isoforms of mammalian PKC are not stimulated by PS alone(28, 29). In contrast, this behavior is characteristic of the atypical ζisoform of PKC (28, 30). No additional stimulation was observed byaddition of Ca²⁺, DAG, or phorbol ester (PMA) as a DAG substitute. Thisbehavior is also exclusively characteristic of PKCζ (28, 30).Interestingly, the cys-rich region of PKC1, which is predicted to be aDAG-binding domain, has been reported to interact with Rho1 intwo-hybrid experiments (19). Therefore, Rho1 may replace DAG in theactivation of PKC1.

[0195] This study provides the first example of a PKC isoform whosestimulation by cofactors is dependent on a GTPase. We have identifiedrecently a second role for Rho1 in the maintenance of cell integrity.Specifically, Rho1 is an essential component of the 1,3-β-glucansynthase (GS) complex (see Example 2, infra), the enzyme responsible forconstructing polymers of 1,3-β-glucan in the cell wall. We have foundthat thermal induction of the FKS2 gene, which encodes another componentof the GS (32, 33), is under the control of PKC1 and MPK1. Based onthese findings, we propose the following model. A signal induced byweakness created in the cell wall during growth (and exacerbated at hightemperature) stimulates guanine nucleotide exchange of Rho1 at thegrowth site. The GTP-bound Rho1 stimulates cell wall constructiondirectly by activating GS and indirectly by stimulating PCK1-dependentgene expression in support of this process (FIG. 5).

[0196] D. References For Example 1

[0197] 1. D. E. Levin et al., (1990) Cell 62: 213-224

[0198] 2. I. Herskowitz (1995) Cell 80: 187-197

[0199] 3. D. E. Levin and B. Errede (1995) Curr. Opin. Cell. Biol. 7:197-202

[0200] 4. Y. Kamada et al., (1995) Genes Dev. 9: 1559-1571

[0201] 5. M. Watanabe et al., (1994) J. Biol. Chem. 269: 16829-16836

[0202] 6. B. Antonsson et al., (1994) J. Biol. Chem. 269: 16821-16828

[0203] 7. A. J. Ridley and A. Hall (1992) Cell 70: 389-399

[0204] 8. A. J. Ridley et al., (1992) Cell 70: 401-410

[0205] 9. C. D. Nobes and A. Hall (1995) Cell 81: 53-62

[0206] 10. C. S. Hill et al., (1995) Cell 81: 1159-1170

[0207] 11. M. F. Olson et al., (1995) Science 269: 1270-1272

[0208] 12. A. Minden et al., (1995) Cell 81: 1147-1157

[0209] 13. O. A. Coso et al., (1995) Cell 81: 1137-1146

[0210] 14. A. B. Vojtek and J. A. Cooper (1995) Cell 82: 527-529

[0211] 15. R. Treisman (1995) EMBO J. 14: 4905-4913

[0212] 16. W. Yamochi et al., (1994) J. Cell Biol. 125: 1077-1093

[0213] 17. P. Madaule et al., (1987) PNAS USA 84: 779-783

[0214] 18. H. Qadota et al., (1994)PNAS USA 91: 9317-9321

[0215] 19. H. Nonaka et al., (1995) EMBO J. 14: 5931-5938

[0216] 20. R. S. Sikorski and P. Hieter (1989) Genetics 122: 19-27

[0217] 21. R. C. Cadwell and G. F. Joyce (1992) PCR Meth. Appl. 2: 28-32

[0218] 22. Y. Ohya and D. Botstein (1994) Genetics 138: 1041-1054

[0219] 23. Y. Zheng et al., (1994) J. Biol. Chem. 269: 2369-2372

[0220] 24. M-N. Simon et al., (1995) Nature 376: 702-705

[0221] 25. Z-S. Zhao et al., (1995) Mol. Cell. Biol. 15: 5246-5257

[0222] 26. E. Manser et al., (1994) Nature, 367: 40-46

[0223] 27. U. G. Knaus et al., (1995) Science, 269: 221-223

[0224] 28. Y. Ono et al., (1989) PNAS USA 86: 3099-3103

[0225] 29. D. J. Burns et al., (1990) J. Biol. Chem. 265: 12044-12051

[0226] 30. A. Toker et al., (1994) J. Biol. Chem. 269: 32358-32367 etal.

[0227] 32. P. Mazur et al., (1995) Mol. Cell. Biol. 15: 5671-5681

[0228] 33. S. B. Inoue et al., (1995) Eur. J. Biochem. 231: 845-854

EXAMPLE 2 Identification of Yeast Rho1 GTPase as a Regulatory Subunit of1,3-β-glucan Synthase

[0229] A. Overview

[0230] 1,3-β-glucan synthase is a multi-enzyme complex that catalyzesthe synthesis of 1,3-β-linked glucan, a major structural component ofthe yeast cell wall. Temperature-sensitive mutants in the essentialRho-type GTPase, Rho1, displayed thermolabile glucan synthase activity,which was restored by the addition of recombinant Rho1. Glucan synthasefrom mutants expressing constitutively active Rho1 did not requireexogenous GTP for activity. Rho1 copurified with 1,3-β-glucan synthaseand was found to associate with the Gsc1/Fks1 subunit of this complex invivo. Both proteins were found to reside predominantly at sites of cellwall remodeling. Therefore, it appears that Rho1 is a regulatory subunitof 1,3-β-glucan synthase.

[0231] The cell wall of the budding yeast Saccharomyces cerevisiae isrequired to maintain cell shape and integrity (1). Vegetativeproliferation requires that the cell remodel its wall to accomodategrowth, which during bud formation, is polarized to the bud tip. Themain structural component responsible for the rigidity of the yeast cellwall is 1,3-β-linked glucan polymers with some branches through1,6-β-linkages. The biochemistry of the yeast enzyme catalyzing thesynthesis of 1,3-β-glucan chains has been studied extensively (2,3), butlittle is known at the molecular level about the genes encoding subunitsof this enzyme. Only a pair of closely related proteins (Gsc1/Fks1 andGsc2/Fks2) are known to be subunits of the 1,3-β-glucan synthase (GS)(3-5). GS activity in many fungal species, including S. cerevisiae,requires GTP or a non-hydrolyzable analog (eg. GTPγS) as a cofactor,suggesting that a GTP-binding protein stimulates this enzyme (2,6). Inthis report, we demonstrate that the Rho1 GTPase is an essentialregulatory component of the GS complex.

[0232] The Saccharomyces RHO1 (Ras homologous) gene encodes a smallGTPase that resides at sites of growth (7), and whose function isessential for viability (M. S. Boguski et al. (1992) New Biol. 4:408).Based on phenotypic analyses of conditional rho1 mutants, we and othershave suggested that the normal function of Rho1 is to maintain cellintegrity (7,9). Conditional rho1 mutants are hypersensitive toCalcofluor white and echinocandin B, drugs that interfere with cell wallassembly, suggesting that this gene is involved in wall construction(10). To determine if Rho1 is required for glucan synthesis, we measuredGS activity in extracts of temperature-sensitive rho1 mutants grown atpermissive temperature. GS activity from wild-type cells increased as afunction of assay temperature from 23° C. to 30° C. to 37° C. (FIG. 6A).All of the rho1 mutants tested displayed reduced levels of activity ateach temperature relative to wild-type. Moreover, the enzyme from allbut one mutant (rho1-5) exhibited some level of thermolability,suggesting that RHO1 function is required for GS activity. Therefore, wetested the ability of purified, recombinant glutathione-S-transferase(GST)-Rho1 to restore GS activity to membrane fractions from the mostimpaired rho1 mutant (rho1-3). Membranes from this mutant were virtuallydevoid of activity at 37° C. FIG. 6B shows that GS activity was restoredfully by the addition of GTPγS-bound GST-Rho1, but not GST-Cdc42,another member of the Rho-family of GTPases. GTPγS could be replacedwith GTP, but not GDP (FIG. 6C). These results indicate that theGS-deficient mutant membranes lack only Rho1 function.

[0233] We also examined GS activity from yeast cells expressing anconstitutively active RHO1 allele (RHO1-Q68H). The analogous mutation inRas results in a protein that is impaired for the ability to hydrolyzeGTP and has transforming potential in mammalian cells (11). The GTPrequirement of GS activity was examined in membranes obtained fromrho1-3 cells overexpressing RHO1 or RHO1-Q68H under the induciblecontrol of the GAL1 promoter (FIG. 7). Under inducing conditions(galactose), expression of RHO1-Q68H resulted in GS activity that wasindependent of exogenous GTP. By contrast, GS activity in membranes fromcells overexpressing RHO1 was largely dependent on GTP. Similar resultswere obtained with another activated allele (RHO1-G19V; 12). Takentogether, these results indicate that GS activity requires functionalRho1 in the GTP-bound state.

[0234] To determine if Rho1 is a component of the GS complex, wemonitored the levels of Rho1 during purification of GS activity. Theenzyme was purified by successive product entrapments followingextraction from membranes (3). FIG. 8 shows that both Rho1 and Gsc1/Fks1were enriched in the partially purified GS. The specific activity of GSwas increased approximately 700-fold through purification, whereas Rho1was enriched approximately 400-fold. GS purified from the rho1-5 mutantwas deficient in GS activity despite normal levels of Rho1 and Gsc1/Fks1proteins (data not shown). To determine if Rho1 copurifies with GSbecause it physically associates with the GS complex, the partiallypurified enzyme was immunoprecipitated with either of two monoclonalantibodies against Gsc1/Fks1. The resultant immunoprecipitates wereanalyzed by SDS-PAGE and immunoblotting with anti-Rho1 antibody. FIG. 9Ashows that Rho1 coimmunoprecipitates with Gsc1/Fks1.

[0235] Finally, we examined the localization of Rho1, tagged at itsNH₂-terminus with the influenza hemagglutinin (HA) epitope (^(HA)Rho1),and Gsc1/Fks1 in growing yeast cells. Rho1 is known to be located at thebud tip (the site of polarized growth) during bud formation, and at themother/bud neck (the site of septum formation) during cytokinesis (7).Indirect immunofluorescence of cells double labeled with anti-HA andanti-Gsc1/Fks1 antibodies revealed that Gsc1/Fks1 colocalizes with^(HA)Rho1 (FIG. 9B). These results strongly suggest that Rho1, likeGsc1/Fks1, is a component of the GS complex. This complex isredistributed through the cell cycle so as to reside at sites of cellwall remodeling.

[0236] We have shown recently that Rho1 interacts with and activates thePKC1 protein kinase (see Example 1, supra). Like rho1 mutants, pkc1mutants display cell integrity defects that result from a deficiency incell wall construction. However, several observations indicate that PKC1is not involved in the activation of GS. First, mutants in PKC1 displayno defect in GS activity (14). Second, overexpression of PKC1 did notrestore GS activity to rho1 mutants (15). Third, PKC1 was not detectedin the purified GS complex (16). Therefore, we propose that Rho1 playsat least two distinct regulatory roles in the maintenance of cellintegrity. One is the activation of GS and the other is the stimulationof PKC1 for signal transduction. Rho1 may serve to coordinate, bothspacially and temporally, several events required for effective cellwall remodeling. Both the GTP requirement for GS activity, and thestructure of fungal PKCs are evolutionarily conserved (6,17), suggestingthat the dual function of Rho1 may be conserved as well.

[0237] C. References and Notes For Example 2

[0238] 1. V. J. Cid et al., (1995) Microbiol. Rev. 59:345; F. M. Klis,(1994) Yeast 10:851

[0239] 2. P. C. Mol et al., (1994) J. Biol. Chem. 269:31267

[0240] 3. S. B. Inoue et al., (1995) Eur. J. Biochem. 231:845

[0241] 4. C. M. Douglas et al., (1994) PNAS USA 91:12907; A. F. J. Ramet al.,(1995) FEBS Lett. 358:165; P. Garett-Engele et al., (1995) Mol.Cell. Biol. 15:4103

[0242] 5. P. Mazur et al., ibid, p. 5671.

[0243] 6. P. J. Szaniszlo et al., (1985) J. Bacteriol. 161:1188

[0244] 7. W. Yamochi et al., (1994) J. Cell. Biol. 125:1077

[0245] 8. P. Madaule et al., (1987) PNAS USA 84:779

[0246] 9. H. Qadota et al., (1994) PNAS USA 91:9317

[0247] 10. Yeast strains YOC752 (rho1-2), YOC729 (rho1-3), YOC754(rho1-4), YOC755 (rho1-5) and YOC764 (wild-type) were used in thisstudy. YOC752, YOC729, and YOC755 displayed hypersensitivity toCalcofluor white and echinocandin B at 23° C.

[0248] 11. C. J. Der et al., (1986) Cell 44:167

[0249] 12. YPH499 cells carrying plasmids with wild-type RHO1 (pYO762),RHO1-G19V (pYO906) under the control of the GAL1 promoter, or vectoralone (pYO761) were used. Cells were incubated in galactose medium for10 h, and GS activity associated with the membrane fraction was measured(3). Most of the GS activity from cells with pYO906 wasGTPγS-independent, whereas only 15-20% of the activity wasGTPγS-independent in the control strains.

[0250] 14. A temperature-sensitive pkc1 strain (SYT11-12A) and itsisogenic wild-type strain (YS3-6D) [S. Yoshida et al., (1992) Mol. Gen.Genet. 231:337] were grown in YPD (yeast extract/peptone/dextrose) at23° C. A pkc1Δ strain (DL376) and its isogenic wild-type (DL100) [D. E.Levin and E. Bartlett-Heubusch, (1992) J. Cell Biol. 116:1221] weregrown at 23° C. in YPD containing 10% sorbitol. GS activities wereassayed at 23° C. and at 37° C.

[0251] 15. Mutants used were rho1-3 and rho1-5 carrying PKC1 on amulticopy plasmid (pYO910), or vector alone (pYO324).

[0252] 16. Partially purified enzyme fraction (second productentrapment) was analyzed by immunoblotting with anti-PCK1 antibody (S.Yoshida, unpublished).

[0253] 17. T. Toda, et al., (1993) EMBO J. 12: 1987; G. Paravicini etal., Yeast, in press.

[0254] 18. Crude yeast extracts were prepared as described [Y. Kamada etal., (1995) Genes Dev. 9:1559], and stored at −80° C. in lysis buffersupplemented with 33% glycerol. Membrane fractions, where indicated,were obtained from crude extracts and 1,3-β-glucan synthase (GS)activity was measured as described in (2) with the followingmodifications: UDP-[³H]glucose was used as the substrate and α-amylase(1U/40 μl) was added to reaction mixtures to eliminate the contributionof [³H]glucose incorporation into glycogen. For all GS assays, the meanand standard error for four experiments is shown.

[0255] 19. Recombinant GST-Rho1 and GST-Cdc42 were expressed in Sf9insect cells, and purified as described previously [Y. Zheng et al.,(1994) J. Biol. Chem. 269:2369].

[0256] 20. A series of protein sample dilutions was analyzed byimmunoblotting with guinea pig anti-Rho1 antiserum or mouseanti-Gsc1/Fks1 monoclonal antibodies (T2B8; 3). The amount of antigenswas estimated by densitometry.

[0257] 21. Goat anti-mouse IgG-agarose (20 μl; Sigma) was incubated with500 μl media from monoclonal antibody cultures for 5 h at 37° C. Theagarose beads were washed 5 times with phosphate-buffered saline andtwice with Buffer A (0.4 CHAPS, 0.08% cholesteryl hemisuccinate, 50 mMTris-Cl, pH 7.5, 1 mM EDTA, 8 μM GTPγS and 33% glycerol). Partiallypurified GS (1.8 μg) was added and the reaction mixtures were furtherincubated for 2 h at 37° C. After washing the beads four times withBuffer A, the bound complexes were analyzed by immunoblotting withanti-Rho1 antiserum or anti-Gsc1/Fks1 monoclonal antibodies (T2B8).

[0258] 22. Cells of haploid strain YOC785, which bears a rho1Δ and theHA-tagged RHO1 gene (13) on a centromere plasmid (pYO904) were doublestained with mouse monoclonal antibody against Gsc1/Fks1 (T2B8) andrabbit anti HA-antibody (Boehringer), as described previously [J. R.Pringle et al., (1989) Methods Cell Biol., 31:357]. Secondary antibodieswere FITC-conjugated anti-mouse IgG (Cappel) and TRITC-conjugatedanti-rabbit IgG (Cappel). Control strains (YPH499 for ^(HA)Rho1 andgsc1Δ for Gsc1/Fks1) produced no signals in single staining experiments.The secondary antibodies did not cross-react with the heterologousprimary antibodies. Some internal punctate staining of Gsc1/Fks1 thatdid not colocalize with ^(HA)Rho1 may represent secretory intermediates.

EXAMPLE 3 Yeast Geranylgeranyl Protein Transferase I is Essential forMembrane Localization of Rho1 GTPase and 1,3-β-glucan Synthase Activity

[0259] The abbreviations used in Example 3 are: GGPTase I,geranylgeranyl protein transferase I; GST, glutathone-S-transferase; HA,influenza hemagglutinin; ORF open reading frame; GS, 1,3-β-glucansynthase.

[0260] A. Overview

[0261] Protein prenylation, farnesylation and geranylgeranylation, is aposttranslational reaction which requires the covalent attachment of ahydrophobic tail, isoprenoid (C15 or C20), to the C-terminal cysteineresidue of the substrate proteins (1). Prenylation is necessary for manyproteins to interact with membranes and to locate at properintracellular places. Many lines of evidence have been accumulated toshow that small GTPases require prenylation to gain full functionality(1, 2).

[0262] Genes encoding subunits of each prenyltransferase have beencloned in the yeast Saccharomyces cerevisiae. The genes CAL1 (3) (alsoknown as CDC43 (4)) and DPR1 (5) (also known as RAM1) encode β subunitsof the yeast GGPTase I and FTase, respectively, and RAM2 encodes thecommon α subunit (6). The α subunit, β subunit and component A of theyeast GGPTase II are encoded by BET4, BET2 and MSI4, respectively (7).An alignment of the homologous regions of the three β subunit sequences(positions 159-350 of the Cal1/Cdc43 sequence) reveals 32-40% identityeach other (3). This region contains novel repeat motifs .(M. S. Boguskiet al. (1992) New Biol. 4:408). The repeats have a length of 44-45residues and there are three repeats in the Cal1p/Cdc43p sequence. Therepeats are conserved in the central Gly-Gly-Phe-Gly-Gly sequenceregion. The α subunit of isoprenyl transferases also possesses distinctinternal repetitive sequence containing tryptophan. Hydrophobic bondsbetween the side chains of the conserved tryptophan and phenylalaninemay be important for forming heterodimer.(M. S. Boguski et al. (1992)New Biol. 4:408).

[0263] Among prenyltransferase mutants, a mutation in the GGPTase I βsubunit gene was the first to be isolated and characterized. cal1-1 wasidentified originally as a mutation resulting in a Ca²⁺ -dependentphenotype.(9). The cal1-1 mutant simultaneously exhibits a homogeneousterminal phenotype with a G2/M nucleus and a small bud at 37° C. (9).Independent screening of yeast cell cycle mutants which accumulatedenlarged unbudded cells identified six other alleles, cdc43-2˜cdc43-7(10). Yeast GGPTase I is essential for yeast cell growth, sincedeletions of the CAL1 gene result in a lethal phenotype (3). However,GGPTase I is no longer essential, when the dosage of the two GTPases,Rho1p (11, 12) and Cdc42p (13), are artificially elevated (14). Sincethe yeast GGPTase I prenylates these two GTPases, Cdc42p and Rho1p areimplicated genetically as the only two essential substrates of GGPTase I(14). CAL1/CDC43 is necessary not only for the function of the smallGTPases but also for membrane localization of the small GTPases. Anincrease in soluble Cdc42p is observed in the cdc43-2 strain grown atthe restrictive temperature (15).

[0264] This study was undertaken to understand the molecular lesionscaused by loss of the GGPTase I function, using the seventemperature-sensitive mutations in the CAL1/CDC43 gene. All of themutation sites were determined at the nucleotide level. An increase insoluble Rho1p was observed in the cal1-1 strain grown at the restrictivetemperature. Futhermore, GS activity was dramatically reduced in thecal1-1 mutant strains. Several phenotypic differences were observedamong the cal1/cdc43 mutations, possibly due to the alteration ofsubstrate specificity caused by the mutations.

[0265] B. Experimental Procedures

[0266] Materials.—YPD medium contained 1% Bacto-yeast extract (DifcoLaboratories, Detroit, Mich.), 2% polypeptone (Nihon Chemicals, Osaka),and 2% glucose (Wako Chemicals, Tokyo). YPD supplemented with 100 mM or300 mM CaCl₂ was used as Ca²⁺-rich medium. Other standard media aredescribed elsewhere (16).

[0267] DNA manipulation—DNA fragments containing the cdc43 mutationswere cloned by gap repair (17). The pCAL-F9 plasmid containing the 2.8kb SphI-PstI fragment of the CAL1/CDC43 gene was digested withNsp(7524)V and EcoT22I and introduced into the cdc43 strains(cdc43-2˜cdc43-7). Transformation of the plasmid containing theNsp(7524)V-EcoT22I gap resulted in repair of the gap to yield plasmidsin which the gap was repaired by gene conversion with the chromosomalsequences. The gap-repaired plasmids were recovered from yeast, and itsNsp(7524)V-EcoT22I fragment was subcloned into the Nsp(7524)V-EcoT22Igap of pCAL-F9. Then, the resulting plasmids YCpT-cdc43-2˜YCpT-cdc43-7were introduced into the cdc43 strains. Because the transformants showeda temperature-sensitive phenotype, we concluded that all of the cdc43mutations resided within the region between the Nsp(7524)V and EcoT22I.Nucleotide sequencing of the 1.0-kb Nsp(7524)V-EcoT22I fragment from theYCpT-cdc43-2˜YCpT-cdc43-7 revealed that each of the cdc43 mutantspossessed a single base pair change within the ORF.

[0268] Production of the anti-Rho1p antibody—The purified GST-Rho1p(64-209) which is a fusion protein of GST with Rho1p from amino acidpositions 64 to 209 was minced and emulsified with R-700 (RIBIImmunoChem Research, Hamilton, Mont.) and the resulting emulsion wasused to immunize four guinea pigs. After boost was repeated five timeswith three-weeks intervals, blood was collected from the animals and oneof the immune serum was used in this study. The anti-Rho1p antibodyspecifically recognized Rho1p. Western blotting analysis showed thatthere was no other protein band detected in the lysates of cellsexpressing human rhoA in place of RHO1.

[0269] Cell Fractionation Experiments. Cell fractionation experimentswere performed using techniques described by Ziman et al. (15). Briefly,cells were grown at 23° C. to mid log phase, and approximately 5×10⁸cells were collected, washed with water, resuspended in 0.1 ml of lysisbuffer (0.8 M sorbitol, 1 mM EDTA, 10 mMN-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid pH 7.0) with 0.5mM PMSF, and lysed on ice by vortexing with 400-500 mm acid-washed glassbeads (Sigma). Greater than 80% lysis was verified by light microscopy.After addition of 0.4 ml of lysis buffer, cell lysates were spun at 390×g for 1 min at 4° C. The supernatant was then spun at 436,000× g for 20min at 4° C., and the pellets were resuspended in the same volume oflysis buffer. To assess the relative amount of Rho1p and Cdc42p in eachfraction, equal volumes of each fraction were loaded onto a sodiumdodecyl sulfate-12.5% polyacrylamide gel for immunoblot analysis. Guineapig polyclonal antibody against Rho1p and mouse monoclonal antibodyagainst HA (12CA5, Boeringer Mannheim, Germany) were used at 1:500 and1:100, respectively. Alkaline phosphatase-conjugated goat anti-guineapig IgG and anti-mouse IgG were used at 1:5000. Antibody-antigencomplexes were detected with 5-bromo-4-chloro-3-indoryl-phosphate andnitro blue tetrazolium.

[0270] C. Results and Discussion

[0271] Mutation points of cdc43-2˜cdc43-7 were determined after DNAfragments containing the cdc43-2˜cdc43-7 mutations were cloned by thegap repair method.(17) to yield YCpT-cdc43-2˜YCpT-cdc43-7. Based on thesubcloning analysis (see Materials and Methods), we concluded that allof the cdc43 mutations resided within the 1.0-kb region between theNsp(7524)V and EcoT22I, nearly corresponding to the entire coding regionof CAL1/CDC43. Nucleotide sequencing of the 1.0-kb Nsp(7524)V-EcoT22Ifragment from the YCpT-cdc43-2˜YCpT-cdc43-7 revealed that each of thecdc43 mutants possessed a single base pair change within the ORF. FIG.10 shows the amino acid changes in the cdc43 sequences. cdc43-4 andcdc43-6 resulted from an identical nucleotide change, and hereafter arereferred to as cdc43-6. cdc43-5 had a amino acid change at the sameposition as cdc43-4 and cdc43-6, but resulted in a different amino acidchange. FIG. 10 shows that the four cdc43/cal1 mutations (cdc43-5cdc43-6, cdc43-7, cal1-1) were mapped within the domain homologous tothe b-subunits of other protein isoprenyltransferases (a.a. position159-350). Interestingly enough, these mutations affect the conservedamino acid residues among the subunits of GGPTase I from four differentspecies (3, 18, 19). The other two cdc43 mutations (cdc43-2 and cdc43-3)were mapped outside of the homologous domain.

[0272] We have previously shown the functional interaction between RHO1and CAL1 based on the observation that overproduction of Rho1psuppressed the temperature sensitivity of cal1-1 (See reference ofExample 3). In order to know whether the suppression by overproductionof Rho1p was seen only with the cal1-1 allele, we examined the abilityof overproduction of Rho1p to suppress the cdc43 mutations. Since therestrictive temperatures of the cdc43 mutants were different, effects ofthe Rho1p overexpression were examined at five different temperatures(23° C., 28° C., 30° C., 33° C. and 37° C.). We found that the cdc43mutations were not suppressed effectively by overproduction of Rho1p(Table 1). None of the mutations was suppressed at 37° C., while cal1-1was suppressed at this temperature. cdc43-2 and cdc43-7 with multicopyRHO1 grew slightly faster than those with vector alone at 30° C., whilecal1-1 was suppressed completely at this temperature. Slight growthimprovement of cdc43-5 by overproduction of Rho1p was observed only at23° C. These results indicate that among the cal1/cdc43 mutations so farisolated, cal1-1 is a unique mutation that is effectively suppressed byoverproduction of Rho1p. TABLE 1 Effect of overproduction of Rho1p andCdc42p in the cal1/cdc43 mutants growth on YPD YPD +Ca strain plasmid23° C. 28° C. 30° C. 33° C. 37° C. 33° C. 37° C. cal1-1 pYO324 + + ± — —++ + YCpT-CAL1 ++ ++ ++ ++ ++ ++ ++ YEpT-RHO1 ++ ++ ++ ++ + ++ +YEpT-CDC42 n.d. n.d. n.d. n.d. n.d. n.d. n.d. cdc43-2 pYO324 ++ + ± — —— — YCpT-CAL1 ++ ++ ++ ++ ++ ++ ++ YEpT-RHO1 ++ + + — — — — YEpT-CDC42++ + ± — — — — cdc43-3 pYO324 ++ ++ ++ ± — — — YCpT-CAL1 ++ ++ ++ ++ ++++ ++ YEpT-RHO1 ++ ++ ++ ± — — — YEpT-CDC42 ++ ++ ++ ± — + — cdc43-5pYO324 + ± — — — — — YCpT-CAL1 ++ ++ ++ ++ ++ ++ ++ YEpT-RHO1 ++ ± — — —— — YEpT-CDC42 ++ ++ ++ ++ + ++ cdc43-6 pYO324 ++ + ± — — — — YCpT-CAL1++ ++ ++ ++ ++ ++ ++ YEpT-RHO1 ++ + ± ± — — — YEpT-CDC42 ++ + ± — — — —cdc43-7 pYO324 ++ + ± — — ± — YCpT-CAL1 ++ ++ ++ ++ ++ ++ ++ YEpT-RHO1++ + + — — ± — YEpT-CDC42 ++ + ± — — + —

[0273] Since overproduction of Rho1p suppressed a mutation of theCAL1/CDC43 gene, we next attempted to examine multicopy suppression ofthe cdc43 mutations by overproduction of another essential substrate ofGGPTase I, Cdc42p. We found that overproduction of Cdc42p suppressed thetemperature-sensitive phenotype of cdc43-5 (Table 1); the cdc43-5 mutantwith multiple copies of CDC42 grew well at 37° C. Among the cdc43mutations, cdc43-5 was most effectively suppressed by overproduction ofCdc42p; cdc43-3, cdc43-6 and cdc43-7 were suppressed slightly byoverproduction of Cdc42p at the intermediate temperature, and cdc43-2was not suppressed at all at any temperature examined.

[0274] Several trials to introduce multiple copies of CDC42 into thecal1-1 strain were unsuccessful. Reasoning that overexpression of Cdc42pmight be a lethal event in the cal1-1 strain, we attempted to increasethe levels of Cdc42p by placing its expression under the control of theGAL1 promoter that was induced by galactose in the medium. The cal1-1strain with pGAL-CDC42 could grow on solid media containing glucose butdid not grow on media containing galactose (FIG. 11). This growthinhibition was observed at any temperature examined (23° C., 30° C. and37° C.). Since pGAL-CDC42 was not toxic in the wild-type strain and manyof the other cdc43 mutants (FIG. 11), we concluded that lethality causedby the overexpression of Cdc42p is specific to the cal1-1 mutant.Although CDC42 on a multicopy plasmid is not toxic in cdc43-7,pGAL-CDC42 is dereterious in cdc43-7 (FIG. 11). This may be due to thefact that the expression level of Cdc42p by pGAL-CDC42 is more than thatexpressed by multiple copies of CDC42.

[0275] cal1-1 was suppressed most effectively by overexpression ofRho1p, while cdc43-5 was suppressed by overexpression of Cdc42p. To testthe possibility that the allele-specific suppression is due to substratespecificity of the mutant GGPTase I, we examined the partitioning ofRho1p and Cdc42p in the cal1-1 and cdc43-5 mutant strains. It wasalready shown that soluble Cdc42p increases in the cdc43-2 strain grownat the restrictive temperature (15), suggesting that membranelocalization of small GTPases is dependent on geranylgeranylmodification. We found that the proportion of Rho1p found in the solublefraction of cal1-1 dramatically increases after the temperature shift(FIG. 12). Rho1p from cdc43-5 strain grown at 37° C. for 2 hr was almostexclusively in the particulate fraction, indicating that increase ofsoluble Rho1p is specific to cal1-1. The proportion of HA-tagged Cdc42pfound in the soluble fraction of cdc43-5 increased after 2 hr incubationat 37° C., while cal1-1 did not affect partitioning of HA-tagged Cdc42p(FIG. 12). Temperature-shift itself did not affect the partitioning ofthese GTPases in the wild-type control strain. These results suggestedthat cal1-1 and cdc43-5 specifically impair geranylgeranylation of Rho1pand Cdc42p, respectively.

[0276] We have previously shown that Rho1p is a regulatory subunit of1,3-β-glucan synthase (see Example 2 above). To directly examineinvolvement of GGPTase I in the Rho1 function, we measured GS activityin membrane fractions of the cal1-1 and cdc43-5 mutant cells grown atpermissive temperature (FIG. 13). We found that cal1-1 displayeddramatically reduced activity relative to wild-type. cdc43-5 mutantinstead displayed only slightly reduced activity, probably due to thefact that cdc43-5 impairs geranylgeranylation of Cdc42p more thangeranylgeranylation of Rho1. We tested whether purified, recombinantGST-Rho1 restored GS activity to the membrane fraction of the cal1-1mutant. GS activity was restored by the addition of constitutivelyactivated Rho1 . These results indicate that the GS-deficient cal1-1mutant membrane lack the Rho1 function.

[0277] Multiple copies of either Rho1p or Cdc42p suppressed specificalleles of cal1/cdc43 (Table 2): cal1-1 was suppressed effectively bymulticopy RHO1, while cdc43-5 was suppressed effectively by multicopyCDC42. Given both Rho1p from the cal1-1 strain and Cdc42p from thecdc43-5 strain accumulate in the soluble fraction, substrate specificipyof the mutant GGPTase I likely accounts for the allele-specificsuppression. In our current model, cal1-1 and cdc43-5 selectively impairthe in vivo geranylgeranylation of Rho1p and Cdc42p, respectively. Thisis consistent with observation of the mutant phenotypes; terminalphenotypes of cdc43-5 and cdc42 are undistinguished, and those of cal1-1and temperature-sensitive rho1 strains are somewhat similar. This isalso consistent with our observation that overexpression of Cdc42p islethal specifically in the cal1-1 strain, because overexpression ofCdc42p likely sequesters the cal1-1 GGPTase I to further impairgeranylgeranylatikn of Rho1p. GS activity was dramatically reduced incal1-1 but not in cdc43-5. Taken together, our genetic and biochemicalresults suggest that the CAL1/CDC43 GGPTase I has an ability toprenylate the substrate GTPases by some domain-specific,substrate-specific recognition mechanisms. TABLE 2 Summary of the effectof the GTPases in the cal1/cdc43 mutants overproduction Phenotype Cdc42pRho1p suppression cdc43-5 cal1-1 (cdc43-3, -4, -7) (cdc43-2, -5, -7)deleterious cal1-1 (cdc43-7)

[0278] C. References in Example 3

[0279] 1. W. R. Schafer and J. Rine (1992) Annu. Rev. Genet. 26:209; S.Clarke (1992) Annu. Rev. Biochem. 61:355

[0280] 2. C. A. Omer and J. B. Gibbs (1994) Mol. Microbiol. 11:219

[0281] 3. Y. Ohya et al. (1991) J. Biol. Chem. 266:12356

[0282] 4. D. I. Johnson et al. (1991) Gene 98:149

[0283] 5. L. E. Goodman et al. (1988) Yeast 4:271

[0284] 6. B. He et al. (1991) Proc. Natl. Acad. Sci. USA 88:11373

[0285] 7. K. Fujimura et al. (1994) J. Biol. Chem. 269:9205; G. Rossi etal. (1991) Nature 351:158

[0286] 8. M. S. Boguski et al. (1992) New Biol. 4:408

[0287] 9. Y. Ohya et al. (1984) Mol. Gen. Genet. 193:389

[0288] 10. A. E. M. Adams et al. (1990) J. Cell. Biol. 111:131

[0289] 11. P. Madaule et al. (1987) Proc. Natl. Acad. Sci. USA 84:779

[0290] 12. H. Qadota et al. (1994) Proc. Natl. Acad. Sci. USA 91:9317

[0291] 13. D. I. Johnson and J. R. Pringle (1990) J. Cell. Biol. 111:143

[0292] 14. Y. Ohya et al. (1993) Mol. Biol. Cell 4:1017

[0293] 15. M. Ziman et al. (1993) ibid. 1307

[0294] 16. M. Rose et al. (1990) Methods in yeast genetics. A laboratorymanual. CSH Lab. Press, CSH, NY.

[0295] 17. T. L. Orr-Weaver et al. (1983) Methods in Enzymol. 101:228

[0296] 18. M. Diaz et al. (1993) EMBO J. 12:5245

[0297] 19. F. L. Zhang et al. (1994) J. Biol. Chem. 269:3175

[0298] 20. H. Qadota et al. (1992) Yeast 8:735

[0299] 22. Inoue et al. (1995) Eur. J. Biochem. 231: 845

[0300] All of the above-cited references and publications are herebyincorporated by reference.

Equivalents

[0301] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, numerous equivalents to thespecific polypeptides, nucleic acids, methods, assays and reagentsdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the following claims.

1 31 4 amino acids amino acid <Unknown> linear peptide 1 Cys Xaa Xaa Xaa1 4 amino acids amino acid <Unknown> linear peptide 2 Xaa Xaa Cys Cys 14 amino acids amino acid <Unknown> linear peptide 3 Xaa Cys Xaa Cys 1 11amino acids amino acid <Unknown> linear protein 4 Thr Glu Asn Thr ValIle Ser Gly Phe Val Gly 1 5 10 13 amino acids amino acid <Unknown>linear protein 5 Lys Glu Ser Lys Gly Ile Lys Tyr Ser Gly Phe Gln Ala 1 510 13 amino acids amino acid <Unknown> linear protein 6 Glu Asp Arg SerAsn Leu Asp Arg Cys Gly Phe Arg Gly 1 5 10 13 amino acids amino acid<Unknown> linear protein 7 Glu Asp Arg Ser Asn Leu Asn Arg Cys Gly PheArg Gly 1 5 10 13 amino acids amino acid <Unknown> linear protein 8 AlaArg Phe Val Ser Lys Cys Gln Arg Pro Asp Arg Gly 1 5 10 12 amino acidsamino acid <Unknown> linear protein 9 Lys Asn Phe Val Glu Leu Cys LysThr Ser Gln Gly 1 5 10 11 amino acids amino acid <Unknown> linearprotein 10 Ala Gly Leu Arg Ala Leu Gln Leu Glu Asp Gly 1 5 10 11 aminoacids amino acid <Unknown> linear protein 11 Ala Gly Leu Arg Ala Leu GlnLeu Glu Asp Gly 1 5 10 13 amino acids amino acid <Unknown> linearprotein 12 Leu Arg Phe Cys Tyr Ile Ala Val Ala Ile Leu Tyr Ile 1 5 10 8amino acids amino acid <Unknown> linear protein 13 Met Arg Gln Leu TyrMet Ala Thr 1 5 8 amino acids amino acid <Unknown> linear protein 14 MetArg Phe Val Tyr Cys Ala Ser 1 5 8 amino acids amino acid <Unknown>linear protein 15 Met Arg Phe Val Tyr Cys Ala Ser 1 5 13 amino acidsamino acid <Unknown> linear protein 16 Asp Gly Gly Phe Gln Gly Arg GluAsn Lys Phe Ala Asp 1 5 10 13 amino acids amino acid <Unknown> linearprotein 17 Ser Gly Gly Leu Asn Gly Arg Thr Asn Lys Asp Val Asp 1 5 10 13amino acids amino acid <Unknown> linear protein 18 Gln Asn Gly Tyr HisGly Arg Pro Asn Lys Pro Val Asp 1 5 10 13 amino acids amino acid<Unknown> linear protein 19 Gln Asn Gly Tyr His Gly Arg Pro Asn Lys ProVal Asp 1 5 10 13 amino acids amino acid <Unknown> linear protein 20 GlnLys Thr Leu Thr Gly Gly Phe Ser Lys Asn Asp Glu 1 5 10 12 amino acidsamino acid <Unknown> linear protein 21 Gln His Ala Leu Gly Gly Phe SerLys Thr Pro Gly 1 5 10 12 amino acids amino acid <Unknown> linearprotein 22 Asp Arg Leu Val Gly Gly Phe Ala Lys Trp Pro Asp 1 5 10 12amino acids amino acid <Unknown> linear protein 23 Asp Arg Leu Val GlyGly Phe Ala Lys Trp Pro Asp 1 5 10 5 amino acids amino acid <Unknown>linear peptide 24 Gly Cys Ile Ile Leu 1 5 7 amino acids amino acid<Unknown> linear peptide 25 Lys Leu Lys Cys Ala Ile Leu 1 5 15 aminoacids amino acid <Unknown> linear peptide 26 Gly Gly Leu His Arg His GlyThr Ile Ile Asn Arg Lys Glu Glu 1 5 10 15 29 base pairs nucleic acidsingle linear other nucleic acid /desc = “primer” 27 CCATCGATCATATGTGTCAA GCTAGGAAT 29 33 base pairs nucleic acid single linear othernucleic acid /desc = “primer” 28 GCGGGTACCC TGCAGTCAAA AACAGCACCT TTT 3342 base pairs nucleic acid single linear other nucleic acid /desc =“primer” 29 GGTAGCTTGA VACATCAAAA CTCCTCCTGC AGATTTATTT TG 42 5 aminoacids amino acid <Unknown> linear peptide 30 Gly Cys Val Ile Ala 1 5 4amino acids amino acid <Unknown> linear peptide 31 Cys Val Ile Ala 1

We claim:
 1. An assay for identifying compounds having potential anti-fungal activity, comprising: (a) forming a reaction mixture including: (i) a fungal geranylgeranyl transferase (GGPTase), (ii) a GGPTase substrate, and (iii) a test compound; and (b) detecting interaction of the GGPTase substrate with the GGPTase, wherein a statistically significant decrease in the interaction of the GGPTase substrate and GGPTase in the presence of the test compound, relative to the level of interaction in the absence of the test compound, indicates a potential anti-fungal activity for the test compound.
 2. The assay of claim 1, wherein GGPTase substrate comprises target polypeptide comprising a fungal Rho-like GTPase, or a polypeptide portion thereof including at least one of (a) a prenylation site which can be enzymatically prenylated by the GGPTase, or (b) a GGPTase binding sequence which specifically binds the GGPTase.
 3. The assay of claim 1, wherein the reaction mixture is a prenylation system including an activated geranylgeranyl group, and the step of detecting the interaction of the GGPTase substrate with the GGPTase comprises detecting conjugation of the geranylgeranyl group to the GGPTase substrate.
 4. The assay of any of claims 3 or 23, wherein at least one of the geranylgeranyl group and the GGPTase substrate comprises a detectable label, and the level of geranylgeranyl group-conjugated to the GGPTase substrate is quantified by detecting the label in at least one of the GGPTase substrate, the geranylgeranyl group, and geranylgeranyl-conjugated GGPTase substrate.
 5. The assay of claim 1, wherein the step of detecting the interaction of the GGPTase substrate with the GGPTase comprises detecting the formation of complexes including the GGPTase substrate with the GGPTase.
 6. The assay of claim 5, wherein at least one of the GGPTase and the GGPTase substrate comprises a detectable label, and the level of GGPTase/GGPTase substrate complexes formed in the reaction mixture is quantified by detecting the label in at least one of the GGPTase substrate, the GGPTase, and GGPTase/GGPTase substrate complexes.
 7. The method of any of claims 4 or 6, wherein the label group is selected from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
 8. The assay of claim 4, wherein the substrate target comprises a fluorescent label, the fluorescent characterization of which is altered by the level of prenylation of the substrate target.
 9. The assay of claim 8, wherein the substrate target comprises a dansylated peptide substrate of the fungal GGPTase.
 10. The assay of any of claims 3 or 23, wherein conjugation of the geranylgeranyl group to the GGPTase substrate is detected by an immunoassay.
 11. The assay of claim 5, wherein the formation of protein-protein complexes including the GGPTase substrate with the GGPTase is detected by an immunoassay.
 12. The assay of any of claims 1 or 23, wherein the reaction mixure is reconstituted protein mixture.
 13. The assay of any of claims 1 or 23, wherein the reaction mixure comprises a cell lysate.
 14. The assay of any of claims 2 or 23, wherein the fungal Rho-like GPTase is selected from the group consisting of Rho1, Rho2, Rsr1/Bud1 and Cdc42, and homologs thereof.
 15. The assay of claim 1, wherein the reaction mixture is a whole cell comprising heterologous nucleic acid recombinantly expressing one or more of the fungal GGPTase subunits and GGPTase substrate.
 16. The assay of claim 1, wherein the reaction mixture is a whole cell comprising a heterologous reporter gene construct comprising a reporter gene in operable linkage with a transcriptional regulatory sequence sensitive to intracellular signals transduced by interaction of the GGPTase substrate and GGPTase.
 17. The assay of any of claims 1, 23, 24, 25, or 26 wherein the assay is repeated for a variegated library of at least 100 different test compounds.
 18. The assay of any of claims 1, 23, 24, 25, or 26 wherein the test compound is selected from the group consisting of small organic molecules, and natural product extracts.
 19. The assay of any of claims 2 or 23, wherein one or more of the GGPTase and target polypeptide are derived from a human pathogen which is implicated in mycotic infection.
 20. The assay of claim 19, wherein the mycotic infection is a mycosis selected from a group consisting of candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, penicilliosis, conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis, maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and sporotrichosis.
 21. The assay of claim 19, wherein the human pathogen is selected from a group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor pusillus.
 22. The assay of claim 19, wherein the human pathogen is Pneumocystis carinii.
 23. An assay for identifying compounds having potential anti-fungal activity, comprising: (a) forming a cell-free reaction mixture including: (i) a fungal geranylgeranyl transferase (GGPTase), (ii) a target polypeptide comprising a fungal Rho-like GTPase, or a polypeptide portion thereof including a prenylation site (iii) an activated geranylgeranyl group, (iv) a divalent cation, and (v) a test compound; (b) detecting conjugation of the gernaylgernayl group of the target polypeptide in the reaction mixture, wherein a statistically significant decrease in the prenylation of the target polypeptide and GGPTase in the presence of the test compound, relative to the level of prenylation in the absence of the test compound, indicates a potential anti-fungal activity for the test compound.
 24. A method of identifying an agent which disrupts the ability of an geranylgeranyl protein transferase (GGPTase) to bind to a fungal GTPase, comprising: i. providing an interaction trap system including (a) a first fusion protein comprising at least a portion of a fungal GGPTase subunit, (b) second fusion protein comprising at least a portion of a fungal GTPase, and (c) a reporter gene, including a transcriptional regulatory sequence sensitive to interactions between the GGPTase portion of the first fusion protein and the GTPase portion of the second polypeptide; ii. contacting the interaction trap system with a candidate agent; iii. measuring the level of expression of a reporter gene in the presence of the candidate agent; and iv. comparing the level of expression of the reporter gene in the presence of the candidate agent to a level of expression in the absence of the candidate agent, wherein a decrease in the level of expression of the reporter gen in the presence of the candidate agent is indicative of an agent that inhibits interaction of the GGPTase and GTPase.
 25. An assay for identifying compounds having potential anti-fungal activity, comprising: (i) providing a first recombinant cell including a first prenylation substrate derived from a Rho-like GTPase which is a substrate for a geranylgeranyl transferase expressed by the cell; (ii) providing a second recombinant cell including a second prenylation substrate identical to the first prenylation substrate except that it is mutated to be a substrate for a farnesyl transferase expressed by the recombinant cell; (iii) contacting the first and second cells with a candidate agent; and (iv) comparing the level of prenylation of the Rho-like GTPases in first and second cells, wherein a statistically significant decrease in the prenylation of the first prenylation substrate, relative to the level of prenylation of the second prenylation substrate, is indicative of an agent that inhibits interaction of a GGPTase and GTPase.
 26. An assay for screening test compounds to identify agents which modulate the interaction of a fungal geranylgeranyl transferase (GGPTase) with a fungal Rho0-like GTPase, comprising: i. providing a cell expressing a recombinant form of one or more of a fungal GGPTase and a fungal Rho-like GTPase; ii. contacting the cell with a test compound; and iii. detecting the level of interaction of the GGPTase and Rho-like GTPase, wherein a statistically significant change in the level of interaction of the GGPTase and Rho-like GTPase is indicative of an agent that modulates the interaction of those two proteins.
 27. The method of claim 23, wherein one or both of a GGPTase subunit or the Rho-like GTPase are fusion proteins.
 28. The method of claim 23, wherein the level of interaction of the GGPTase and Rho-like GTPase is detected by detecting prenylation of the Rho-like GTPase.
 29. The method of claim 25, wherein the Rho-like GTPase is a fusion protein further comprising a transcriptional regulatory protein, and level of prenylation of the Rho-like GTPase is detected by measuring the level of expression of a reporter gene construct which is sensitive to the transcriptional regulatory protein portion of the fusion protein, wherein inhibition of prenylation of the fusion protein results in loss of membrane partitioning of the fusion protein and increases expression of the reporter gene construct.
 30. The assay of any of claims 1, 20, 21, 22, or 23, which comprises a further step of preparing a pharmaceutical preparation of one or more compounds identified as having potential antifungal activity.
 31. An assay for identifying compounds having potential antifungal activity, comprising: i. forming a reaction mixture including a fungal Rho-like GTPase, a fungal protein kinase C (PKC), and a test compound; and ii. detecting interaction of the Rho-like GTPase and PKC, wherein a statistically significant decrease in the interaction of the Rho-like GTPase and PKC in the presence of the test compound, relative to the level of interaction in the absence of the test compound, indicates a potential antifungal activity for the test compound.
 32. The assay of claim 31, wherein the reaction mixture is a kinase system including ATP and a PKC substrate, and the step of detecting interaction of the GTPase and PKC comprises detecting phosphorylation of the PKC substrate by a PKC/GTPase complex.
 33. The assay of claim 32, wherein at least one of the PKC substrate and ATP comprises a detectable label, and the level of phosphorylation of the PKC substrate is quantified by detecting the label in at least one of the PKC substrate or ATP.
 34. The assay of claim 31, wherein the step of detecting the interaction of the GTPase with the PKC comprises detecting the formation of protein-protein complexes including the GTPase and PKC.
 35. The assay of claim 34, wherein at least one of the PKC and GTPase comprises a detectable label, and the level of PKC/GTPase complexes formed in the reaction mixture is quantified by detecting the label in at least one of the GTPase, the PKC, and PKC/GTPase complexes.
 36. The method of any of claims 33 or 35, wherein the label group is selected from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
 37. The assay of claim 36, wherein the detectable label is a protein having a measurable activity, and one of the PKC or GTPase is fusion protein including the detectable label.
 38. The assay of claim 33, wherein the PKC substrate comprises a fluorescent label, the fluorescent characterization of which is altered by the level of phosphorylation of the PKC substrate.
 39. The assay of claim 33, wherein phosphorylation of the PKC substrate is detected by immunoassay.
 40. The assay of claim 34, wherein the formation of protein-protein complexes including the GTPase and PKC is detected by an immunoassay.
 41. The assay of claim 31, wherein the reaction mixure is reconstituted protein mixture.
 42. The assay of claim 31, wherein the reaction mixure comprises a cell lysate.
 43. The assay of claim 31, wherein the GPTase is selected from the group consisting of Rho1, Rho2, Rsr1/Bud1 and Cdc42, and fungal homologs thereof.
 44. The assay of claim 31, wherein the reaction mixture is a whole cell comprising heterologous nucleic acid recombinantly expressing one or more of the PKC and GTPase.
 45. The assay of claim 31, wherein the reaction mixture is a whole cell comprising a heterologous reporter gene construct comprising a reporter gene in operable linkage with a transcriptional regulatory sequence sensitive to intracellular signals transduced by interaction of the GTPase and PKC.
 46. The assay of claim 31, wherein the assay is repeated for a variegated library of at least 100 different test compounds.
 47. The assay of claim 31, wherein the test compound is selected from the group consisting of small organic molecules, and natural product extracts.
 48. The assay of claim 31, wherein one or more of the PKC and GTPase are derived from a human pathogen which is implicated in mycotic infection.
 49. The assay of claim 48, wherein the mycotic infection is a mycosis selected from a group consisting of candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, penicilliosis, conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis, maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and sporotrichosis.
 50. The assay of claim 48, wherein the human pathogen is selected from a group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillusfumigatus, Aspergillusflavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor pusillus.
 51. The assay of claim 48, wherein the human pathogen is Pneumocystis carinii.
 52. The assay of claim 31, which comprises a further step of preparing a pharmaceutical preparation of one or more compounds identified as having potential antifungal activity.
 53. An assay for identifying compounds having potential antifungal activity, comprising: i. forming a reaction mixture including a fungal Rho-like GTPase, a fungal glucan synthase complex or subunit thereof (GS protein), and a test compound; and ii. detecting interaction of the Rho-like GTPase and GS protein, wherein a statistically significant decrease in the interaction of the Rho-like GTPase and GS protein in the presence of the test compound, relative to the level of interaction in the absence of the test compound, indicates a potential antifungal activity for the test compound.
 54. The assay of claim 53, wherein the reaction mixture is a glucan synthesis system including a GTP and a UDP-glucose, and the step of detecting interaction of the GTPase and GS protein comprises detecting formation of glucan polymers in the reaction mixture.
 55. The assay of claim 54, wherein the UDP-glucose comprises a detectable label, and the level of glucan polymer formation is quantified by detecting the labelled glucan polymers.
 56. The assay of claim 53, wherein the step of detecting the interaction of the GTPase with the GS protein comprises detecting the formation of protein-protein complexes including the GTPase and GS protein.
 57. The assay of claim 56, wherein at least one of the GS protein and GTPase comprises a detectable label, and the level of GS protein/GTPase complexes formed in the reaction mixture is quantified by detecting the label in at least one of the GTPase, the GS protein, and GS protein/GTPase complexes.
 58. The method of any of claims 53 or 57, wherein the label group is selected from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
 59. The assay of claim 56, wherein the formation of protein-protein complexes including the GTPase and GS protein is detected by an immunoassay.
 60. The assay of claim 53, wherein the reaction mixure is reconstituted protein mixture.
 61. The assay of claim 53, wherein the reaction mixure comprises a cell lysate.
 62. The assay of claim 53, wherein the GPTase is selected from the group consisting of Rho1, Rho2, Rsr1/Bud1 and Cdc42, and fungal homologs thereof.
 63. The assay of claim 53, wherein the reaction mixture is a whole cell comprising heterologous nucleic acid recombinantly expressing one or more of the GS protein and GTPase.
 64. The assay of claim 53, wherein the assay is repeated for a variegated library of at least 100 different test compounds.
 65. The assay of claim 53, wherein the test compound is selected from the group consisting of small organic molecules, and natural product extracts.
 66. The assay of claim 53, wherein one or more of the GS protein and GTPase are derived from a human pathogen which is implicated in mycotic infection.
 67. The assay of claim 66, wherein the mycotic infection is a mycosis selected from a group consisting of candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, penicilliosis, conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis, maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and sporotrichosis.
 68. The assay of claim 66, wherein the human pathogen is selected from a group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramrosa, and Mucor pusillus.
 69. The assay of claim 66, wherein the human pathogen is Pneumocystis carinii.
 70. The assay of claim 53, which comprises a further step of preparing a pharmaceutical preparation of one or more compounds identified as having potential antifungal activity.
 71. A recombinant cell comprising (i) exogenous nucleic acid encoding one or more subunits of a fungal geranylgeranyl protein transferase (GGPTase), and (ii) exogenous nucleic acid encoding a fungal Rho-like GTPase or a fragment thereof including at least one of (a) a prenylation site which can be enzymatically prenylated by the GGPTase, or (b) a GGPTase binding sequence which specifically binds the GGPTase.
 72. The cell of claim 71, wherein one or more of the nucleic acids encoding the GGPTase and GTPase are derived from a human pathogen which is implicated in mycotic infection.
 73. The cell of claim 72, wherein the mycotic infection is a mycosis selected from a group consisting of candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, penicilliosis, conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis, maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and sporotrichosis.
 74. The cell of claim 72, wherein the human pathogen is selected from a group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor pusillus.
 75. The cell of claim 72, wherein the human pathogen is Pneumocystis carinii.
 76. The cell of claim 71, which cell is a recombinantly manipulate yeast cell selected from the group consisting of Kluyverei spp, Schizosaccharomyces spp, Ustilaqo spp and Saccharomyces spp.
 77. The cell of claim 71, which cell is a recombinantly manipulate Schizosaccharomyces cerivisae cell.
 78. The cell of claim 71, which cell is constitutively or inducibly defective for an endogenous activity corresponding to one or more of the GGPTase and GTPase encoded by the exogenous nucleic acids.
 79. A reconstituted protein mixture or a cell lysate mixture comprising (i) a recombinant fungal geranylgeranyl protein transferase (GGPTase), and (ii) a recombinant fungal Rho-like GTPase or a fragment thereof including at least one of (a) a prenylation site which can be enzymatically prenylated by the GGPTase, or (b) a GGPTase binding sequence which specifically binds the GGPTase.
 80. The mixture of claim 79, wherein one or more of the recombinant GGPTase and GTPase are derived from a human pathogen which is implicated in mycotic infection.
 81. The mixture of claim 80, wherein the mycotic infection is a mycosis selected from a group consisting of candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, penicilliosis, conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis, maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and sporotrichosis.
 82. The mixture of claim 80, wherein the human pathogen is selected from a group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramrosa, and Mucor pusillus.
 83. The mixture of claim 80, wherein the human pathogen is Pneumocystis carinii.
 84. A recombinant cell comprising (i) exogenous nucleic acid encoding a fungal Rho-like GTPase, and (ii) exogenous nucleic acid encoding a fungal protein selected from the group consisting of a fungal protein kinase C (PKC) or one or more subunits of a fungal glucan synthase.
 85. A reconstituted protein mixture or a cell lysate mixture comprising (i) a recombinant fungal Rho-like GTPase, and (ii) a recombinant fungal protein selected from the group consisting of a fungal protein kinase C (PKC) or a fungal glucan synthase.
 86. A recombinant cell comprising exogenous nucleic acid encoding one or more subunits of a geranylgeranyl protein transferase (GGPTase) cloned from a human fungal pathogen.
 87. A recombinant cell comprising exogenous nucleic acid encoding a Rho-like GTPase cloned from a human fungal pathogen.
 88. A recombinant cell comprising exogenous nucleic acid encoding one or more subunits of a glucan synthase cloned from a human fungal pathogen.
 89. A recombinant cell comprising exogenous nucleic acid encoding a protein kinase C cloned from a human fungal pathogen.
 90. A reconstituted protein mixture or a cell lysate mixture comprising one or more subunits of a recombinant geranylgeranyl protein transferase (GGPTase) cloned from a human fungal pathogen.
 91. A reconstituted protein mixture or a cell lysate mixture comprising a recombinant Rho-like GTPase cloned from a human fungal pathogen.
 92. Areconstituted protein mixture or a cell lysate mixture comprising one or more recombinantn subunits of a glucan synthase cloned from a human fungal pathogen.
 93. A reconstituted protein mixture or a cell lysate mixture comprising a recombinant protein kinase C cloned from a human fungal pathogen.
 94. The cell of any of claims 86-93 wherein the human pathogen is selected from a group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor pusillus. 